Compositions and methods for increasing cholesterol efflux and raising hdl using atp binding cassette transporter protein abc1

ABSTRACT

The present invention relates to novel ABC1 polypeptides and nucleic acid molecules encoding the same. The invention also relates to recombinant vectors, host cells, and compositions comprising ABC1 polynucleotides, as well as to methods for producing ABC1 polypeptides. The invention also relates to antibodies that bind specifically to ABC1 polypeptides. In addition, the invention relates to methods for increasing cholesterol efflux as well as to methods for increasing ABC1 expression and activity. The present invention further relates to methods for identifying compounds that modulate the expression of ABC1 and methods for detecting the comparative level of ABC1 polypeptides and polynucleotides in a mammalian subject. The present invention also provides kits and compositions suitable for screening compounds to determine the ABC1 expression modulating activity of the compound, as well as kits and compositions suitable to determine whether a compound modulates ABC1-dependent cholesterol efflux.

This application is a divisional of U.S. application Ser. No.10/920,989, filed Aug. 18, 2004 which is a divisional of U.S.application Ser. No. 09/596,141 filed Jun. 16, 2000, now U.S. Pat. No.6,821,774, issued Nov. 23, 2004, which claims benefit of U.S.Provisional Application No. 60/140,264 filed Jun. 18, 1999, U.S.Provisional Application No. 60/153,872 filed Sep. 14, 1999 and U.S.Provisional Application No. 60/166,573 filed Nov. 19, 1999, each ofwhich is incorporated herein by reference in its entirety.

TECHNICAL FIELD OF INVENTION

The present invention relates to novel ABC1 polypeptides and nucleicacid molecules encoding the same. The invention also relates torecombinant vectors, host cells, and compositions comprising ABC1polynucleotides, as well as to methods for producing ABC1 polypeptides.The invention also relates to antibodies that bind specifically to ABC1polypeptides. In addition, the invention relates to methods forincreasing cholesterol efflux as well as to methods for increasing ABC1expression and activity. The present invention further relates tomethods for identifying compounds that modulate the expression of ABC1and methods for detecting the comparative level of ABC1 polypeptides andpolynucleotides in a mammalian subject. The present invention alsoprovides kits and compositions suitable for screening compounds todetermine the ABC1 expression modulating activity of the compound, aswell as kits and compositions suitable to determine whether a compoundmodulates ABC1-dependent cholesterol efflux.

BACKGROUND OF THE INVENTION

Circulating lipids in human plasma or lymphatic fluid consist ofcholesterol, cholesteryl esters, triglycerides and phospholipids. Theselipids are transported in large molecular complexes called lipoproteins,which consist of a core of cholesteryl esters and/or triglycerides, anenvelope of phospholipids and free cholesterol, and apolipoproteins(Scriver et al., Eds., The Metabolic and Molecular Basis of InheritedDisease, 7^(th) Ed., p. 1841-1851 (McGraw-Hill, New York 1995)).Apolipoproteins are involved in the assembly and secretion of thelipoprotein, as well as the activation of lipoprotein modifying enzymes,such as lecithin cholesterol acyl transferase (LCAT). In addition,apolipoproteins provide structural integrity and are ligands for a largespectrum of receptors and membrane docking proteins. The plasmalipoproteins are categorized into five types according to size:chylomicrons (largest in size and lowest in density), very low densitylipoproteins (VLDL), intermediate density lipoproteins (IDL), lowdensity lipoproteins (LDL) and high density lipoprotein (HDL).

Chylomicrons, VLDLs, IDLs, and LDLs transport exogenous and endogenouscholesterol and triacylglycerols to peripheral sites, where the lipidsplay a role in various metabolic pathways and serve as a majorconstituent of cell membranes. Chylomicrons are assembled in theintestinal mucosa as a means to transport dietary cholesterol andtriacylglycerols to various tissues. VLDLs are formed in the liver totransport endogenous cholesterol and triacylglycerols synthesized by theliver to extra-hepatic tissues, such as muscle and adipose tissue. Infasting serum, VLDLs contain 10-15% of the total serum cholesterol andmost of the triglyceride. In circulation, VLDLs are converted to LDLsthrough the action of lipoprotein lipase. LDLs are the primary plasmacarriers of cholesterol for delivery to all tissues, typicallycontaining 60-70% of the total fasting serum cholesterol.

In contrast, HDLs are involved in “reverse cholesterol transport”, thepathway by which excess cholesterol is transported from peripheral sitesback to the liver, where it is excreted in the form of bile salts(Glomset, J. A., J. Lipid Res., 9, 155-167 (1968)). Nascent HDLs aresynthesized de novo in the liver and small intestine, as protein-richdisc-shaped particles devoid of cholesterol and cholesterol esters. Infact, a major function of HDLs is to act as circulating stores ofapolipoproteins, primarily apo C-I, apo C-II, and apoE. The nascent orprotein-rich HDLs are converted into spherical lipoprotein particlesthrough the accumulation of cholesteryl esters obtained from cellularsources. The HDL normally contain 20-30% of the total fasting serumcholesterol.

According to current theories, the reverse efflux of cellularcholesterol to HDL is mediated through two mechanisms: an aqueousdiffusion pathway and an apolipoprotein-mediated pathway. The relativeimportance of these distinguishable mechanisms depends on the cell typeand metabolic state (Oram et al., J. Lipid Res., 37:2743-2491 (1996);Rothblat et al., J. Lipid Res., 40:781-796 (1999); Stein et al.,Atherosclerosis, 144:285-301 (1999)). For many cells, the aqueousdiffusion pathway is the principle pathway through which cholesterolefflux occurs (Johnson et al., Biochim. Biophys. Acta, 1085:273-298(1991)). This pathway involves the bidirectional exchange of cholesterolbetween cell membranes and a lipoprotein acceptor, such as HDL, in theextracellular space through a process of passive transport (Remaley etal., Arterioscler. Thromb. Vasc. Biol., 17:1813-1821 (1997); Rothblat etal., J. Lip. Res., 40:781-796 (1999)). The exchange may occur primarilyat surface microdomains known as caveolae (Fielding et al.,Biochemistry, 34:14288-14292 91995)). Net efflux can be driven byconversion of cholesterol in the extracellular compartment tocholesteryl ester by the action of LCAT.

Alternatively, in macrophage and fibroblast cells, cholesterol andphospholipid efflux is primarily mediated through apolipoproteins, suchas apo A-I, apo A-II, and Apo E (Remaley, supra (1997); Francis, et al.,J. Clin. Invest., 96:78-87 (1995); Vega et al., J. Intern. Med.,226:5-15 (1989); Sakar et al., Biochim. Biophys. Acta, 1438: 85-98(1999); Hara et al., J. Biol. Chem., 266:3080-3086 (1991); Fielding etal., J. Lipid Res., 38, 1503-1521 (1997); Oram et al., J. Lipid Res.,37, 2743-2491 (1996)). The process of apolipoprotein-mediated lipidefflux particularly dominates in macrophages and other scavenger cellswhen they are cholesterol-loaded and/or growth-arrested.Apolipoprotein-mediated efflux is an active transport process thatrequires the direct interaction of the apolipoprotein with the cellsurface, the lipidation of the apolipoprotein, and the subsequentdissociation of the lipid-apolipoprotein particle from the cell (Oram,supra (1996); Mendez, A. J., J. Lipid Res., 38, 1807-1821 (1997);Remaley, supra (1997); Mendez, A. J., J. Lipid Res., 37, 2510-2524(1996)). Once removed from the cell, the cholesterol-rich HDL particlesare transported to the liver and removed from the body as described.

Abnormal lipoprotein function and/or metabolism resulting from geneticdefect or as a secondary effect of another disorder can have seriousbiological consequences. In addition to dietary influences, disorderssuch as diabetes, hypothyroidism, and liver disease can result inelevated plasma levels of LDL-cholesterol and triglycerides. Elevatedlevels of LDL-cholesterol and triglycerides have been identified asmajor risk factors associated with the incidence of coronary heartdisease, which is the primary cause of death in the United States andother industrialized nations (Hokanson et al., J. Cardiovasc. Risk.,3:213-219 (1996); The Expert Panel, JAMA, 269:3015-3023 (1993)). Theaccumulation of excess LDL-cholesterol on arterial walls can lead to theformation of atherosclerotic plaques, which play a major role in thedevelopment of heart disease. A plaque is believed to form when freeradicals released from arterial walls oxidize LDL. According to theory,the oxidized form of LDL triggers an inflammatory response, attractingcirculating cells to the site which contribute to the formation of alipid plaque. Among these are macrophages and other cells that containscavenger receptors that accumulate cholesterol in an unregulated manner(Brown et al., Ann. Rev. Biochem., 52:223-261 (1986)). Vast stores ofinternal cholesterol result in conversion to a foam cell phenotype,which is believed to be a major contributor to the development ofvascular lesions. As the plaque builds up, the arterial walls constrict,reducing blood flow to the heart.

Interestingly, however, an estimated 60% of heart attacks occur inpersons who do not have elevated blood levels of LDL-cholesterol. Ofthese, an estimated 45% are associated with below average blood levelsof HDL-cholesterol, indicating that low HDL-cholesterol level is asignificant risk factor for coronary heart disease. In fact, recentstudies have indicated that a decreased HDL-cholesterol level is themost common lipoprotein abnormality seen in patients with prematurecoronary artery disease (Genest J., Circulation, 85:2025-2033 (1992);Genest et al., Arterioscler. Thromb., 13:1728-1737 (1993)). Although thebasis for the inverse association between HDL-cholesterol and coronaryheart disease is not well understood, it has been suggested that thecardioprotective role of HDL may stem from its activity relating to thepromotion of cholesterol efflux from macrophage foam cells inatherosclerotic lesions.

One example of cardiovascular disease associated with low HDL is Tangierdisease (TD), a rare genetic disorder characterized by a near orcomplete absence of circulating HDL. In addition to near zero plasmalevels of HDL, patients with TD have a massive deposition andaccumulation of cholesteryl esters in several tissues, includingtonsils, lymph nodes, liver, spleen, thymus, intestine, and Schwanncells (Fredrickson, D. S., J. Clin. Invest., 43, 228-236 (1964); Assmannet al., The Metabolic Basis of Inherited Disease, (McGraw-Hill, NewYork, 1995)). Although the cellular mechanisms have not been previouslyidentified, recent studies have shown that cells from subjects with TDare defective in the process of apolipoprotein-mediated removal ofcholesterol and phospholipids (Remaley et al., Arterioscler. Thromb.Vasc. Biol., 17, 1813-1821 (1997); Francis et al., J. Clin. Invest., 96,78-87 (1995); Rogler et al., Arterioscler. Thromb. Vasc. Biol., 15,683-690 (1995)). These results have led to the proposal that the severeHDL deficiency in TD patients stems from the inability of nascent apoA-I to acquire lipids. Because they do not mature into lipid-richparticles, the nascent HDL in TD patients is rapidly catabolized andremoved from the plasma, resulting in the near zero levels ofcirculating HDL (Remaley, supra (1997); Francis, supra (1995); Horowitzet al., J. Clin. Invest., 91, 1743-1752 (1993); Schaefer et al., J. Lip.Res., 22:217-228 (1981)).

Other disorders associated with severe premature atherosclerosis andhigh risk for coronary heart disease resulting from diminishedHDL-cholesterol levels are hypoalphalipoproteinemia and familial HDLdeficiency syndrome (FHA). Persons with these disorders often havenormal LDL-cholesterol and triglyceride levels. In addition, disorderssuch as diabetes, alcoholism, hypothyroidism, liver disease, andelevated blood pressure can result in diminished plasma levels ofHDL-cholesterol, although many of these disorders are also accompaniedby elevated LDL-cholesterol and triglceride levels.

Current treatments for coronary heart disease have focused primarily ondiet manipulations and/or drug therapies aimed at lowering the plasmalevel of LDL-cholesterol by inhibiting LDL secretion or promoting LDLturnover. Derivatives of fibric acid, such as clofibrate, gemfibrozil,and fenofibrate, promote rapid VLDL turnover by activating lipoproteinlipase. Nicotinic acid reduces plasma levels of VLDL and LDL byinhibiting hepatic VLDL secretion. In addition, HMG-CoaA reductaseinhibitors, such as mevinolin, mevastatin, pravastatin, simvastatin,fluvastatin, and lovastatin reduce plasma LDL levels by inhibiting theintracellular synthesis of cholesterol, which causes an increase in thecellular uptake of LDL. In addition, bile acid-binding resins, such ascholestyrine, colestipol and probucol decrease the level ofLDL-cholesterol by increasing the catabolism of LDL-cholesterol in theliver.

However, many of these therapies are associated with low efficacy and/orside effects that may prevent long-term use. For example, use ofHMG-CoaA reductase inhibitors carry a significant risk of toxicitybecause they inhibit the synthesis of mevalonate, which is required forthe synthesis of other important isoprenoid compounds in addition tocholesterol. Also, gemfibrozil and nicotinic acid are associated withserious adverse effects, including renal injury, myopathy, myoglobinuriaand intolerable skin flushing and itching. In addition, the role ofprobucol in treating patients with coronary heart disease is uncertainbecause its administration results in lower HDL-cholesterol levels as aside effect of reducing LDL-cholesterol.

Furthermore, treating patients who have isolated low HDL-cholesterollevels provides a particularly difficult therapeutic challenge. Forinstance, patients with Tangier disease exhibit a 4- to 6-fold increasein cardiovascular disease even though their LDL levels are alreadyreduced by about 50%. While there is some evidence that gemfibrozil andnicotinic acid may simultaneously elevate HDL levels, in general,therapies aimed at lowering plasma LDL-cholesterol levels are noteffective for Tangier patients who suffer from coronary heart disease asa result of diminished HDL levels. Likewise, patients withhypoalphalipoproteinemia, familial HDL deficiency syndrome, or othercardiovascular disease resulting from low levels of HDL will not benefitfrom therapies aimed at lowering the level of plasma LDL.

The problems associated with current therapies for cardiovasculardisease stem partially from the fact that the biology involved in themovement of cholesterol in and out of cells is not fully understood.Furthermore, the proteins that play a role in cholesterol movement arenot fully known. Therefore, there remains a need for a betterunderstanding of cholesterol cell biology, as well as new methods fortreating humans suffering from cardiovascular disease and otherdisorders associated with hypercholesterolemia. Additionally, thereremains a need for new methods of diagnosing cardiovascular disease andnew methods of screening patients to identify those at high risk fordeveloping cardiovascular disease.

The identification of genes and proteins involved in cholesteroltransport would be useful in the development of pharmaceutical agentsfor the treatment of heart disease and other disorders associated withhypercholesterolemia and atherosclerosis. In addition, theidentification of such genes would be useful in the development ofscreening assays to screen for compounds that regulate the expression ofgenes associated with cholesterol transport. The identification of suchregulatory compounds would be useful in the development of furthertherapeutic agents. Furthermore, the identification of genes andproteins involved in cholesterol transport would be useful as diagnosticindicators of cardiovascular disease and other disorders associated withhypercholesterolemia.

SUMMARY OF THE INVENTION

The present invention provides novel polypeptides and polynucleotidesinvolved in cholesterol efflux. Specifically, the present inventionprovides novel ATP-Binding Cassette (ABC1) polypeptides and novelpolynucleotides that encode ABC1 polypeptides. The terms “ABC1” and“ABCA1” are alternative names for the same ATP-Binding Cassette proteinand gene. The invention provides ABC1 polypeptides, polypeptidefragments, and polypeptide variants. In one preferred embodiment, thepresent invention provides an isolated polypeptide comprising SEQ ID NO:2. In another preferred embodiment, the present invention provides anisolated polypeptide comprising an amino acid sequence that has at least98% identity to SEQ ID NO: 2. The present invention also provides ABC1polypeptides from Tangier disease patients. In one preferred embodiment,the present invention provides an isolated polypeptide comprising SEQ IDNO: 8. In another preferred embodiment, the present invention providesan isolated polypeptide comprising SEQ ID NO: 10.

In addition, the present invention provides ABC1 polynucleotides,polynucleotide fragments, and polynucleotide variants. In one preferredembodiment, the present invention provides an isolated polynucleotidethat encodes the polypeptide comprising SEQ ID NO: 2. In anotherpreferred embodiment, the invention provides an isolated polynucleotidethat encodes a polypeptide comprising an amino acid sequence that has atleast 98% identity to SEQ ID NO: 2. Also, in other preferredembodiments, the invention provides an isolated polynucleotidecomprising a nucleotide sequence that is complementary to apolynucleotide encoding the polypeptide comprising SEQ ID NO: 2 or anisolated polynucleotide comprising a nucleotide sequence that iscomplementary to a polynucleotide encoding the polypeptide comprising anamino acid sequence that has at least 98% identity to SEQ ID NO: 2.

In another preferred embodiment, the present invention provides anisolated ABC1 polynucleotide comprising SEQ ID NO: 1. In a furtherpreferred embodiment, the present invention provides an isolatedpolynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1. In yetanother preferred embodiment, the invention provides a polynucleotidecomprising a nucleotide sequence that has at least 90% identity with SEQID NO: 1. More preferably, the polynucleotide comprises a nucleotidesequence that has at least 95% identity with SEQ ID NO: 1. In other morepreferred embodiments, the polynucleotide comprises a nucleotidesequence that has at least 96%, 97%, 98%, or 99% identity to SEQ IDNO: 1. Also, in other preferred embodiments, the present inventionprovides an isolated polynucleotide comprising a nucleotide sequencethat is complementary to the polynucleotide comprising SEQ ID NO: 1, anisolated polynucleotide comprising a nucleotide sequence that iscomplementary to a polynucleotide comprising nucleotides 291-7074 of SEQID NO: 1, and an isolated polynucleotide that is complementary to apolynucleotide comprising a nucleotide sequence that has at least 90%identity with SEQ ID NO: 1.

The present invention also provides ABC1 polynucleotides correspondingto the 5′ flanking region of the ABC1 gene. In one preferred embodiment,the invention provides an isolated polynucleotide comprising SEQ ID NO:3. In other preferred embodiments, the invention provides an isolatedpolynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643,1292-1643, or 1394-1532 of SEQ ID NO: 3. Preferably, the isolatedpolynucleotide comprises nucleotides 1394-1532 of SEQ ID NO: 3. Inanother preferred embodiment, the invention provides an isolatedpolynucleotide that hybridizes under stringent conditions to apolynucleotide comprising SEQ ID NO: 3. Also, in other preferredembodiments, the present invention provides an isolated polynucleotidethat hybridizes under stringent conditions to a polynucleotidecomprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or1394-1532 of SEQ ID NO: 3. In yet another preferred embodiment of thepresent invention, an isolated polynucleotide that has at least 80%identity to a polynucleotide comprising SEQ ID NO: 3 is provided. Morepreferably, the polynucleotide has at least 90% identity to apolynucleotide comprising SEQ ID NO: 3. Even more preferably, thepolynucleotide has at least 95% identity to a polynucleotide comprisingSEQ ID NO: 3. Also provided in preferred embodiments is an isolatedpolynucleotide that has at least 80% identity to a polynucleotidecomprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or1394-1532 of SEQ ID NO: 3. More preferably, the polynucleotide has atleast 90% identity, and even more preferably at least 95% identity, to apolynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643,1292-1643, or 1394-1532 of SEQ ID NO: 3. In addition, the presentinvention provides an isolated polynucleotide comprising a nucleotidesequence that is complementary to the above described 5′ flanking ABC1polynucleotides. In one preferred embodiment, the invention provides anisolated polynucleotide comprising a nucleotide sequence that iscomplementary to a polynucleotide comprising SEQ ID NO: 3. In anotherpreferred embodiment, the present invention provides an isolatedpolynucleotide comprising a nucleotide sequence that is complementary toa polynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643,1292-1643, or 1394-1532 of SEQ ID NO: 3.

The present invention also provides ABC1 polynucleotides correspondingto the 3′ flanking region of the ABC1 gene. In preferred embodiments,the invention provides an isolated polynucleotide comprising SEQ ID NO:4, SEQ ID NO: 5, or SEQ ID NO: 6, and the complementary sequencesthereof. In other preferred embodiments, the invention provides anisolated polynucleotide that hybridizes under stringent conditions to apolynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6,and the complementary sequences thereof. In still other preferredembodiments, the invention provides an isolated polynucleotide that hasat least 80% identity to a polynucleotide comprising SEQ ID NO: 4, SEQID NO: 5, or SEQ ID NO: 6, and the complementary sequence thereof. Morepreferably, the polynucleotide has at least 90% identity to apolynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.Even more preferably, the polynucleotide has at least 95% identity to apolynucleotide comprising SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

In addition, the present invention also provides ABC1 polynucleotidesfrom Tangier disease patients. In one preferred embodiment, the presentinvention provides an isolated polynucleotide encoding the polypeptidecomprising SEQ ID NO: 8. In another preferred embodiment, the presentinvention provides an isolated polynucleotide comprising SEQ ID NO: 7.In yet another embodiment, the present invention provides an isolatedpolynucleotide encoding the polypeptide comprising SEQ ID NO: 10. Instill another preferred embodiment, the present invention provides anisolated polynucleotide comprising SEQ ID NO: 9. The present inventionfurther provides an isolated polynucleotide comprising a nucleotidesequence that is complementary to the described polynucleotides.

In another aspect, the present invention provides a compositioncomprising any of the above described polynucleotides and a suitablecarrier. In one preferred embodiment, the present invention provides acomposition comprising an isolated polynucleotide encoding a polypeptidecomprising SEQ ID NO: 2, a polynucleotide comprising SEQ ID NO: 1, apolynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1, or apolynucleotide encoding a polypeptide comprising an amino acid sequencethat has at least 98% identity to SEQ ID NO: 2, and a suitable carrier.In another preferred embodiment, the composition comprises an isolatedpolynucleotide comprising a nucleotide sequence that has at least 90%identity with a polynucleotide comprising SEQ ID NO: 1 and a suitablecarrier. In other preferred embodiments, the composition comprises anisolated polynucleotide comprising SEQ ID NO: 3 or an isolatedpolynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643,1292-1643, or 1394-1532 of SEQ ID NO: 3 and a suitable carrier. In stillother preferred embodiments, the invention provides a compositioncomprising a polynucleotide that hybridizes under stringent conditionsto a polynucleotide comprising SEQ ID NO: 3, or a polynucleotidecomprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or1394-1532 of SEQ ID NO: 3, as well as a composition comprising apolynucleotide that has at least 80% identity to a polynucleotidecomprising SEQ ID NO: 3, or a polynucleotide comprising nucleotides1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1532 of SEQ ID NO: 3and a suitable carrier. Also provided by the present invention is acomposition comprising an isolated polynucleotide comprising anucleotide sequence that is complementary to any of the describedpolynucleotides and a suitable carrier.

In addition, the present invention provides recombinant vectors and hostcells comprising any of the described ABC1 polynucleotide sequences. Inone preferred embodiment, the present invention provides a recombinantvector comprising an isolated polynucleotide encoding a polypeptidecomprising SEQ ID NO: 2, an isolated polynucleotide comprising SEQ IDNO: 1, an isolated polynucleotide comprising nucleotides 291-7074 of SEQID NO: 1, or an isolated polynucleotide encoding the polypeptidecomprising an amino acid sequence that has at least 98% identity to SEQID NO: 2. In another preferred embodiment, the recombinant vectorcomprises an isolated polynucleotide comprising a nucleotide sequencethat has at least 90% identity, and more preferably at least 95%identity, with a polynucleotide comprising SEQ ID NO: 1. In stillanother preferred embodiment, the recombinant vector comprises anisolated polynucleotide comprising SEQ ID NO: 7 or SEQ ID NO: 9. Thepresent invention further provides a recombinant vector comprising anisolated polynucleotide comprising a nucleotide sequence that iscomplementary to any of the described polynucleotides. In yet anotherpreferred embodiment, the recombinant vector comprises any of thedescribed polynucleotides and further comprises a heterologous promoterpolynucleotide. One suitable heterologous promoter is a cytomegaloviruspromoter. In a particularly preferred embodiment, the recombinant vectoris pCEPhABC1.

The present invention also provides a recombinant vector comprising anisolated polynucleotide comprising an ABC1 5′ flanking sequence. In onepreferred embodiment, the invention provides a recombinant vectorcomprising an isolated polynucleotide comprising SEQ ID NO: 3 or anisolated polynucleotide comprising nucleotides 1-1532, 1080-1643,1181-1643, 1292-1643, or 1394-1532 of SEQ ID NO: 3. In still otherpreferred embodiments, the invention provides a recombinant vectorcomprising a polynucleotide that hybridizes under stringent conditionsto the polynucleotide of SEQ ID NO: 3, or a polynucleotide comprisingnucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1532 of SEQID NO: 3, as well as a recombinant vector comprising a polynucleotidethat has at least 80% identity to these polynucleotides. The presentinvention further provides a recombinant vector comprising an isolatedpolynucleotide comprising a nucleotide sequence that is complementary toany of the described polynucleotides. In yet another preferredembodiment, the recombinant vector comprises any of the describedpolynucleotides and further comprises at least one polynucleotideencoding a heterologous polypeptide. Suitable heterologous polypeptidesinclude luciferase, β-galactosidase, chloramphenicol acetyl transferase,and green fluorescent proteins. Preferably, the heterologous polypeptideis a luciferase protein. In a particularly preferred embodiment, therecombinant vector is pAPR1.

In addition, the present invention provides host cells comprising any ofthe described recombinant vectors. The present invention furtherprovides compositions comprising any of the described recombinantvectors and a suitable carrier.

The present invention also provides methods for producing the ABC1protein in a mammalian host cell as well as methods for expressing theABC1 protein in a mammalian subject. The method for producing an ABC1protein in a mammalian host cell comprises the steps of: (a)transfecting the mammalian host cell with a recombinant expressionvector comprising a polynucleotide encoding ABC1 in an amount sufficientto produce a detectable level of ABC1 protein, and (b) purifying theproduced ABC1 protein. The method for expressing ABC1 protein in amammalian subject comprises the step of administering to a mammaliansubject a recombinant expression vector comprising a polynucleotideencoding ABC1 in an amount sufficient to express ABC1 protein in themammalian subject.

In addition, the present invention provides compositions and methodssuitable for increasing cholesterol efflux from cells of a mammaliansubject. In one preferred embodiment, the method comprises administeringto the mammalian subject a recombinant expression vector comprising apolynucleotide encoding ABC1 in an amount sufficient to increasecholesterol efflux from the cells. Suitable recombinant expressionvectors include vectors comprising an isolated polynucleotide encoding apolypeptide comprising SEQ ID NO: 2, an isolated polynucleotidecomprising SEQ ID NO: 1, an isolated polynucleotide comprisingnucleotides 291-7074 of SEQ ID NO: 1, and an isolated polynucleotideencoding the polypeptide comprising an amino acid sequence that has atleast 98% identity to SEQ ID NO: 2. Preferred expression vectors includeviral vectors, especially adenoviral vectors and lentiviral vectors. Inother embodiments, the invention provides non-viral delivery systems,including DNA-ligand complexes, adenovirus-ligand-DNA complexes, directinjection of DNA, CaPO₄ precipitation, gene gun techniques,electroporation, liposomes and lipofection.

In another preferred embodiment, the method for increasing cholesterolefflux from cells of a mammalian subject comprises administering to themammalian subject a therapeutic amount of a compound that increases theexpression of ABC1 in the cells. One suitable method comprisesadministering to the mammalian subject a cAMP analogue. Suitable cAMPanalogues include 8-bromo cAMP, N6-benzoyl cAMP, and 8-thiomethyl cAMP.Another suitable method comprises administering to the mammalian subjecta compound that increases the synthesis of cAMP, e.g. forskolin. Yetanother suitable method comprises administering to the mammalian subjecta compound that inhibits the degradation of cAMP, such as aphosphodiesterase inhibitor. Suitable phosphodiesterase inhibitorsinclude rolipram, theophylline, 3-isobutyl-1-methylxanthine, R020-1724,vinpocetine, zaprinast, dipyridamole, milrinone, aminone, pimobendan,cilostamide, enoximone, peroximone, and vesnarinone.

In addition, another suitable method for increasing cholesterol effluxfrom cells of a mammalian subject comprises administering to themammalian subject a least one ligand for a nuclear receptor in an amountsufficient to increase cholesterol efflux. Suitable ligands include LXR,RXR, FXR, SXR and PPAR ligands. In one preferred embodiment, the methodcomprises administering to a mammalian subject a ligand for an LXRnuclear receptor. Suitable LXR ligands include 20(S) hydroxycholesterol,22(R) hydroxycholesterol, 24(S) hydroxycholesterol,25-hydroxycholesterol, and 24(S), 25 epoxycholesterol. Preferably, theLXR ligand is 20(S) hydroxycholesterol. In another preferred embodiment,the method comprises administering to a mammalian subject a ligand foran RXR nuclear receptor. Suitable RXR ligands include 9-cis retinoicacid, retinol, retinal, all-trans retinoic acid, 13-cis retinoic acid,acitretin, fenretinide, etretinate, CD 495, CD564, TTNN, TTNNPB, TTAB,and LGD 1069. Preferably, the RXR ligand is 9-cis retinoic acid. Inanother preferred embodiment, the method comprises administering to amammalian subject a ligand for a PPAR nuclear receptor. One suitableligand is a ligand selected from the class of thiazolidinediones. In yetanother preferred embodiment, the method comprises administering atleast two ligands for a nuclear receptor. In a particularly preferredembodiment, the ligands are 20(S) hydroxycholesterol and 9-cis retinoicacid.

In addition, another suitable method for increasing cholesterol effluxfrom cells of a mammalian subject comprises administering to themammalian subject an eicosanoid in an amount sufficient to increasecholesterol efflux. Suitable eicosanoids include prostaglandin E2,prostaglandin J2, and prostacyclin (prostaglandin I2).

In another embodiment, the present invention provides a method forincreasing cholesterol efflux from cells of a mammalian subjectcomprising administering to the mammalian subject a compound thatincreases ABC1 activity in an amount sufficient to increase cholesterolefflux from the cells.

The present invention also provides methods suitable for increasing thegene expression of ABC1 in a mammalian subject. In one preferredembodiment, the method comprises administering to the mammalian subjectat least one ligand for a nuclear receptor in anamount sufficient toincrease the gene expression of ABC1. Suitable ligands include ligandsfor LXR, RXR, FXR, SXR, and PPAR nuclear receptors. In another preferredembodiment, the method comprises administering to the mammalian subjecta cAMP analogue in an amount sufficient to increase the gene expressionof ABC1. In yet another preferred embodiment, the method comprisesadministering to the mammalian subject a compound that increases thesynthesis of cAMP in an amount sufficient to increase the geneexpression of ABC1.

In addition, the present invention provides a method for screening atest compound for ABC1 expression modulating activity comprising thesteps of: (a) operatively linking a reporter cDNA with an expressionmodulating portion of the mammalian ABC1 gene to produce a recombinantreporter construct; (b) transfecting the recombinant reporter constructinto a population of host cells; (c) assaying the level of reporter geneexpression in a sample of the host cells; (d) contacting the host cellswith the test compound being screened; (e) assaying the level ofreporter gene expression in a sample of the host cells after contactwith the test compound; and (f) comparing the relative change in thelevel of reporter gene expression caused by exposure to the testcompound, thereby determining the ABC1 expression modulating activity.The recombinant reporter construct comprises a reporter gene operativelylinked to an expression modulating portion of the mammalian ABC1 gene,such as any of the ABC1 5′ flanking region sequences provided by thepresent invention. In one preferred embodiment, the expressionmodulating portion of the ABC1 gene comprises SEQ ID NO: 3. In anotherpreferred embodiment, the expression modulating portion of the ABC1 genecomprises nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643,1394-1643, or 1394-1532 of SEQ ID NO: 3. Suitable reporter cDNAs includeluciferase, β-galactosidase, chloramphenicol acetyl transferase, andgreen fluorescent protein cDNA. Preferably, the host cell is a mammaliancell. In a particularly preferred embodiment of the method, therecombinant reporter construct is pAPR1.

Also provided by the present invention is a method for screening a testcompound to determine whether the test compound promotes ABC1-mediatedcholesterol efflux from cells in culture comprising the steps of: (a)assaying the level of cholesterol efflux in a sample of mammalian cellsmaintained in culture to determine a control level of cholesterolefflux; (b) contacting the cells with the test compound being screened;(c) assaying the level of cholesterol efflux in a sample of cells aftercontact with the test compound; (d) assaying the level of ABC1-mediatedcholesterol efflux in a sample of cells after contact with the testcompound, thereby determining whether the test compound promotesABC1-mediated cholesterol efflux from cells in culture. The cells can bederived from primary cultures or a cell line. Suitable cells forscreening the test compound include fibroblast, macrophage, hepatic, andintestinal cell lines. Preferably, the cell line is RAW 264.7. In onepreferred embodiment, the ABC1-mediated cholesterol efflux is measuredusing an anti-ABC1 antibody that inhibits the activity of ABC1 uponbinding. In another preferred embodiment, the ABC1-mediated cholesterolefflux is measured using an antisense ABC1 polynucleotide. In aparticularly preferred embodiment, the antisense polynucleotidecomprises SEQ ID NO: 57.

In addition, the present invention provides methods for detecting thecomparative level of ABC1 expression in cells of a mammalian subject.Such methods can be used to determine the susceptibility of a subject tocoronary heart disease. A method for detecting the comparative level ofABC1 expression in cells of a mammalian subject is provided whichcomprises (a) obtaining a cell sample from the mammalian subject, (b)assaying the level of ABC1 mRNA expression in the cell sample; and (c)comparing the level of ABC1 mRNA expression in the cell sample with apre-determined standard level of ABC1 mRNA expression, thereby detectingthe comparative level of ABC1 gene expression in the cells of amammalian subject. Suitable methods for measuring the level of ABC1 mRNAexpression include, for example, RT-PCR, northern blot, and RNAseprotection assay.

The present invention also provides methods for detecting thecomparative level of ABC1 protein in cells of a mammalian subject. Suchmethods can be used to determine the susceptibility of a subject tocoronary heart disease. A method for detecting the comparative amount ofABC1 protein in the cells of a mammalian subject is provided whichcomprises (a) obtaining a cell sample from the mammalian subject, (b)assaying the amount of ABC1 protein in the cell sample, and (c)comparing the amount of ABC1 protein in the cell sample with apre-determined standard amount of ABC1 protein, thereby detecting thecomparative level of ABC1 protein in the cells of the mammalian subject.The amount of ABC1 protein can be determined using various immunoassaysavailable in the art. For example, the amount of ABC1 protein can bedetermined by (a) contacting the cell sample with a population ofanti-ABC1 antibodies and (b) detecting the specific-binding ABC1antibodies associated with the sample. Suitable methods for detectingABC1 antibodies include western blotting, immunoprecipitation, and FACS.

In another aspect, the present invention provides antibodies that bindspecifically to the described ABC1 polypeptides. In one preferredembodiment, the present invention provides an isolated antibody thatbinds specifically to an isolated polypeptide comprising SEQ ID NO: 2.In another preferred embodiment, the invention provides an isolatedantibody that bind specifically to an isolated polypeptide comprising anamino acid sequence that has at least 98% identity with SEQ ID NO: 2.The antibody can be a monoclonal antibody or the antibody can be apolyclonal antibody. In yet another embodiment, the antibody, uponbinding to an ABC1 polypeptide, inhibits the cholesterol transportactivity of the ABC1 polypeptide.

In addition, the present invention provides kits suitable for screeninga compound to determine the ABC1 expression modulating activity of thecompound comprising a reporter cDNA operatively linked to an expressionmodulating portion of the mammalian ABC1 gene in an amount sufficientfor at least one assay and instructions for use. In one preferredembodiment, the kit further comprises means for detecting the reportergene. In another preferred embodiment, the expression modulating portionof the mammalian ABC1 gene comprises SEQ ID NO: 3. In yet anotherpreferred embodiment, the expression modulating portion of the mammalianABC1 gene comprises nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643,1394-1643, or 1394-1532 of SEQ ID NO: 3. Suitable reporter cDNAs includeluciferase, β-galactosidase, chloramphenicol acetyl transferase, andgreen fluorescent protein cDNA. Preferably, the reporter cDNA isluciferase. In a particularly preferred embodiment of the method, therecombinant reporter construct is pAPR1.

The present invention also provides kits suitable for screening acompound to determine whether the compound modulates ABC1-dependentcholesterol efflux. In one preferred embodiment, the kit comprises aninactivating anti-ABC1 antibody in an amount sufficient for at least oneassay and instructions for use. In another preferred embodiment, the kitcomprises an antisense ABC1 oligonucleotide in an amount sufficient forat least one assay and instructions for use. In a particularly preferredembodiment, the antisense ABC1 oligonucleotide comprises SEQ ID NO: 53.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-D is a graphical representation showing the results of controlcholesterol efflux and cholesterol efflux in the presence of HDL and apoA-I from normal fibroblast cells (1A, C) and fibroblast cells fromTangier disease patients (1B, D). The open circles represent thecholesterol efflux from cells that were not exposed to HDL or apo A-I,the closed circles represent the cholesterol efflux from cells exposedto HDL, and the closed diamonds represent the cholesterol efflux fromcells exposed to apo A-I;

FIG. 2 is a graphical representation of a gene expression microarrayanalysis showing a comparison of the gene expression found in cells froma Tangier patient (TD1) and that found in normal cells, whereby a totalof 58,800 human cDNAs were hybridized with cDNA prepared from mRNA ofcAMP-treated TD1 cells cDNA (labeled with Cy3 dye) and with cDNAprepared from mRNA of cAMP-treated normal cells (labeled with Cy5 dye);

FIG. 3 is a schematic diagram showing a restriction map of therecombinant expression vector pCEPhABC1, which contains the open readingframe of the human ABC1 gene;

FIG. 4 is a schematic diagram of the gene structure of human ABC1,showing a comparison between the published human ABC1 amino acidsequence (GenBank, Accession #AJ012376) and the presently disclosed andclaimed human ABC1 amino acid sequence (“CVT”) which has sixtyadditional amino acids at the N-terminal end;

FIG. 5 is a graphical representation showing the inhibitory effect thatABC1 transport inhibitors 4,4-diisothiocyanostilbene-2,2′-disulfonicacid (DIDS) and sulphobromophtaleine (BSP) have on apo A-I-mediatedcholesterol efflux, wherein the open circles indicate the apoA-I-mediated cholesterol in the presence of BSP and the closed circlesindicate the apo A-I-mediated cholesterol in the presence of DIDS;

FIG. 6 is a graphical representation showing the inhibitory effect of anantisense ABC1 oligonucleotide on apo A-I-mediated cholesterol efflux,showing the apo A-I-mediated cholesterol efflux in cells incubatedwithout antisense oligonucleotide, the apo A-I-mediated cholesterolefflux in cells exposed to 30 μM β-globin antisense oligonucleotide, andthe apo A-J-mediated cholesterol efflux in cells exposed to 30 μM ABC1antisense oligonucleotide;

FIG. 7 is a graphical representation demonstrating the stimulation ofapo A-I-mediated cholesterol efflux caused by overexpression of the ABC1gene using RAW 264.7 mouse macrophage cells stably transfected with anexpression plasmid for human ABC1 (pCEPhABC1), showing the apoA-I-mediated cholesterol efflux in control parental cells (no pCEPhABC1)and the apo A-I-mediated cholesterol efflux in clonal cells transfectedwith pCEPhABC1 (L3, L5, L6);

FIG. 8 is a graphical representation of reverse transcription polymerasechain reaction (RT-PCR) analyses showing the level of ABC1 geneexpression in normal cells and cells from Tangier's disease patients(TD1 and TD2) that have been either exposed to albumin (closed bars),exposed to 8-Br-cAMP (open bars), cholesterol-loaded (shaded bars), orcholesterol-loaded and subsequently exposed to apo A-I (hatched bars);

FIG. 9 is a graphical representation of the results of RT-PCR analysesshowing the level of ABC1 gene expression in RAW 264.7 cells exposed toeither ethanol (0.1% v/v), 9-cis retinoic acid (9-cis RA; 10 μM), 20(S)hydroxycholesterol (20(S)-OH; 10 μM), or 9-cis RA and 20(S)-OH (10 μMeach);

FIG. 10 shows the results of immunoprecipitation analyses indicating thelevel of cell-surface ABC1 protein found in normal fibroblasts (NL1,10A) and fibroblasts from a Tangier's disease patient (TD1, 10B) in thepresence of either no additives (control), 8-Br-cAMP (1 mM), cholesterol(30 μg/ml), or cholesterol and 8-Br-cAMP (30 μg/ml and 1 mM,respectively);

FIG. 11 is a schematic diagram showing a restriction map of therecombinant expression vector pAPR1, which contains the 5′ flankingregion of the ABC1 gene positioned upstream of the open reading frame ofthe luciferase reporter gene;

FIG. 12 is a graphical representation showing the level of luciferasereporter gene expression induced in RAW 264.7 cells transfected withpAPR1 in the presence of either EtOH (control), 20(S)-OH (10 μM), 9-cisRA (10 μM), or both 20(S)-OH and 9-cis RA (10 μM each);

FIG. 13 is a schematic diagram of the 5′ flanking region of the ABC1gene.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, novel polypeptides thatincrease the cholesterol efflux from cells are provided. In particular,the present invention provides novel ATP-Binding Cassette 1 (ABC1)polypeptides that have been shown to increase cholesterol efflux.

ABC1 is a member of the family of ATP-binding cassette proteins thatreside in cell membranes and utilize ATP hydrolysis to transport a widevariety of substrates across the plasma membrane. It should be notedthat the terms “ABC1” and “ABCA1” both refer to the same ATP-bindingcassette protein. The term “ABCA1” was introduced in 1999 by anomenclature committee and has received limited acceptance in the field.To date, more than 30 members of this family have been identified in thehuman genome. These homologous proteins contain channel-like structuresthrough which molecules are transported through the cell membrane andone or two domains which bind to ATP to couple energy generatingATP-hydrolysis to transport. Family members include the multidrugresistance factors (MDR/P-glycoproteins; Chen et al., Cell, 47: 381-389(1986); Stride et al., Mol. Pharmacol., 49:962-971 (1996)), transportersassociated with antigen presentation (Neefjes et al., Science,261:769-771 (1993); Shepherd et al., Cell, 74:577-584 (1993)), and thecystic fibrosis transmembrane conductance regulator (Chang et al., J.Biol. Chem., 269:18572-18575 (1994); Rommens et al., Science, 245:1059-1065 (1989)). Members of the ABC transporter family are generallycomposed of 4 domains found within two symmetric halves that are linkedby a long charged region and a highly hydrophobic segment. Each halfcontains a hydrophobic domain, containing 6 transmembrane segments and ahydrophilic nucleotide binding domain containing highly conserved WalkerA and B sequence motifs typical of many ATPases (Hyde et al., Nature,346:362-365 (1990); Luciani et al., Genomics, 21: 150-159 (1994)). Thetransporter activity is dependent on the interaction with ATP at thenucleotide binding domains and by regulation via phosphorylation ofresidues in the region linking the two symmetric halves (Becq et al., J.Biol. Chem., 272: 2695-2699 (1997)).

Several lines of evidence described herein identified ABC1 as a pivotalprotein in the apolipoprotein-mediated mobilization of intracellularcholesterol stores. First, the studies presented herein showed that ABC1is defective in Tangier disease, a genetic disorder characterized byabnormal HDL-cholesterol metabolism. As shown previously, and herein atExample 1, the genetic defect in Tangier disease causes a defect in thepathway of apolipoprotein mediated efflux of cholesterol from withincells, resulting in significantly decreased cholesterol efflux activityand low HDL-cholesterol levels (Oram et al., J. Lipid Res., 37:2743-2491(1996); Francis et al., J. Clin. Invest., 96: 78-87 (1995)). Geneticlinkage analysis of families with Tangier disease assigned the defectivegene to an interval on chromosome 9q31 (Rust et al., Nature Genetics,20: 96-98 (1998)). A search of public databases revealed that the ABC1gene was localized to chromosome 9q22-9q31, which is broader than, butincludes the interval revealed in Rust et al. (Luciani et al., supra(1994)). Based on that data, radiation hybrid mapping of the human ABC1gene was performed, which placed the gene between two markers squarelywithin the 7-cM region of human chromosome 9q31 reported by Rust et al.In addition, as shown in Example 2, microarray analysis revealed thatthe ABC1 gene is 2.5-fold underexpressed in Tangier patient cells ascompared with normal cells. These studies identified the defective genein Tangier disease as ABC1. In addition, further studies presentedherein linked ABC1 activity to cholesterol efflux activity. First,studies showed that inhibitors of ABC1 transport activity, such as4,4-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) andsulphobromophtaleine (BSP), also inhibited apoAI-mediated cholesterolefflux from fibroblast cells (see Example 6). Also, inhibition of ABC1gene expression, using an antisense ABC1 oligonucleotide, was shown toinhibit apoAI-mediated cholesterol efflux from fibroblast cells (Example7). In contrast, transfection studies, in which the ABC1 gene wastransfected into mouse monocyte cells, showed that overexpression ofABC1 results in an increase in apoAI-mediated efflux (Example 8).Finally, RT-PCR performed using wildtype and Tangier patient mRNArevealed that ABC1 mRNA expression is regulated by cellular conditionsrelated to cholesterol efflux in normal skin fibroblast cells, but notin Tangier patient fibroblasts (Example 9). Based on these findings, itwas determined that ABC1 plays a major role in cholesterol efflux.

It is postulated that ABC1 plays a role in the translocation ofintracellular cholesterol to the outer leaflet of the plasma membrane.Deficient transport of intracellular cholesterol due to a lack of ABC1or defective ABC1 results in a lack of cholesterol in specific membranedomains with which apoAI and other apolipoproteins specifically interact(Stangl et al., J. Biol. Chem., 273: 31002-31008 (1998); Babitt et al.,J. Biol. Chem., 272: 13242-13249 (1997))). The failed delivery ofcholesterol to apoAI leads to the formation of cholesterol-deficient HDLparticles that are rapidly removed from the plasma. (Bojanovski et al.,J. Clin. Invest., 80: 1742-1747 (1987)).

DEFINITIONS

The following definitions are provided to facilitate understanding ofcertain terms used throughout this specification.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.

As used herein, the term “polynucleotide(s)” is defined to encompass DNAand RNA of both synthetic and natural origin. The polynucleotide mayexist as single- or double-stranded DNA or RNA, or an RNA/DNAheteroduplex. Thus, the polynucleotide of the present invention can becomposed of any polyribonucleotide or polydeoxyribonucleotide, which maybe unmodified RNA or DNA or modified RNA or DNA. For example,polynucleotides can be composed of single- and double-stranded DNA, DNAthat is a mixture of single- and double-stranded regions, or single,double-, and triple-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or triple-stranded, or a mixture of single-and double-stranded regions. In addition, the polynucleotide can becomposed of triple-stranded regions comprising RNA or DNA or both RNAand DNA. A polynucleotide may also contain one or more modified bases orDNA or RNA backbones modified for stability or for other reasons.“Modified” bases include, for example, tritylated bases and unusualbases such as inosine. A variety of modifications can be made to DNA andRNA; thus, “polynucleotide” embraces chemically, enzymatically, ormetabolically modified forms of polynucleotides. The term“polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

The term “polypeptide(s)” refers to any peptide or protein comprisingtwo or more amino acids joined to each other by peptide bonds ormodified peptide bonds. “Polypeptide” refers to both short amino acidsequences, commonly referred to as peptides, as well as longer aminoacid sequences, generally referred to as proteins. The polypeptide maycontain amino acids other than the 20 gene encoded amino acids.Moreover, the polypeptide may be modified either by natural processes,such as processing and other post-translational modifications, or bychemical modification techniques, which are well-known in the art. Agiven polypeptide may contain many types of modifications. Also, thesame type of modification may be present in the same or varying degreeat one or more sites in the polypeptide. Modifications may occuranywhere in the polypeptide, including the peptide backbone, the aminoacid side-chains, and the amino or carboxyl termini. Modificationsinclude, but are not limited to, acetylation, acylation,ADP-ribosylation, amidation, formylation, gamma-carboxylation,glycosylation, hydroxylation, iodination, methylation, myristoylation,oxidation, phosphorylation, prenylation, sulfation, and selenoylation,as well as the covalent attachment of a nucleotide or nucleotidederivative, a lipid or lipid derivative, or a phosphotidylinositol.Other modifications include cross-linking, cyclization, formation ofpyroglutamate, GPI anchor formation, proteolytic processing,racemization, and t-RNA-mediated addition of amino acids, such asarginylation and ubiquitination. See, for example, Proteins—Structureand Molecular Properties, 2^(nd) Ed., T. E. Creighton, W. H. Freedmanand Co., New York (1993); Wold, F., Posttranslational ProteinModification: Perspectives and Prospects, in Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Meth. Enzymol., 182: 626-646 (1990); and Rattanet al., Protein Synthesis: Posttranslational Modifications and Aging,Ann. N.Y. Acad. Sci., 663: 48-62 (1992)). The polypeptides of theinvention can be prepared in any suitable manner. Such polypeptidesinclude isolated naturally occurring polypeptides, recombinantlyproduced polypeptides, synthetically produced polypeptides, orpolypeptides produced by a combination of these methods. Means forpreparing such polypeptides are well understood in the art.

A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to SEQ ID NO:3 or nucleotides 1-1532, 1080-1643, 1181-1643,1292-1643, 1394-1643, or 1394-1532 of SEQ ID NO: 3, or the complementsthereof. A polynucleotide of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to SEQ ID NO:4, SEQ ID NO: 5, or SEQ ID NO: 6 or thecomplements thereof.

“Stringent hybridization conditions” refers to an overnight incubationat 42° C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

As used herein, the term “complementary” refers to the hybridization orbase pairing between nucleotides, such as, for example, between the twostrands of a double-stranded polynucleotide or between anoligonucleotide primer and a primer binding site on a single-strandedpolynucleotide to be amplified or sequenced. Two single-strandednucleotide molecules are said to be complementary when the nucleotidesof one strand, optimally aligned with appropriate nucleotide insertions,deletions or substitutions, pair with at least about 80% of thenucleotides of the other strand.

“Identity”, as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. “Identity” or “similarity” alsohas an art-recognized meaning that refers to the degree of sequencerelatedness between polypeptide or polynucleotide sequences, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be calculated using a number of well known methods,including those published in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, (1988); Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, (1993); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H. G., eds., Humana Press, New Jersey, (1994); SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, (1987);and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, (1991); and Carillo, H., and Lipton, D., SIAMJ Applied Math 48:1073 (1988)). Methods commonly employed to determineidentity or similarity between two sequences include, but are notlimited to, those disclosed in “Guide to Huge Computers,” Martin J.Bishop, ed., Academic Press, San Diego, (1994), and Carillo, H., andLipton, D., SIAM J Applied Math 48:1073 (1988). Preferred methods todetermine identity are designed to give the largest match between thesequences tested. Methods for aligning polynucleotides or polypeptidesare codified in computer programs, including the GCG program package(Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387 (1984)),BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. 215:403(1990), Bestfit program (Wisconsin Sequence Analysis Package, Version 8for Unix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711 (using the local homology algorithm of Smithand Waterman, Advances in Applied Mathematics 2:482-489 (1981)).

When using any of the sequence alignment programs to determine whether aparticular sequence is, for instance, 90% identical to a referencesequence, the parameters are set so that the percentage of identity iscalculated over the full length of the reference polypeptide orpolynucleotide and that gaps in identity of up to 10% of the totalnumber of nucleotides in the reference polynucleotide are allowed.

A preferred method for determining the best overall match between aquery sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, can bedetermined using the FASTDB computer program based on the algorithm ofBrutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). The term“sequence” includes nucleotide and amino acid sequences. In a sequencealignment the query and subject sequences are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB search of a DNA sequence to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, and Cutoff Score=1, Gap Penalty=5, GapSize Penalty 0.05, and Window Size=500 or query sequence length innucleotide bases, whichever is shorter. Preferred parameters employed tocalculate percent identity and similarity of an amino acid alignmentare: Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty=0.05, and Window Size=500 or query sequence length in amino acidresidues, whichever is shorter.

As an illustration, a polynucleotide having a nucleotide sequence of atleast 90% “identity” to a sequence contained in SEQ ID NO: 1 means thatthe nucleotide sequence of the polynucleotide is identical to a sequencecontained in SEQ ID NO: 1 except that the polynucleotide sequence mayinclude up to ten point mutations per each 100 nucleotides of the totallength of SEQ ID NO: 1. In other words, to obtain a polynucleotidecomprising a nucleotide sequence that has at least 90% identity to SEQID NO: 1, up to 10% of the nucleotides in the sequence contained in SEQID NO: 1 can be deleted, inserted, or substituted with othernucleotides. These changes may occur anywhere throughout thepolynucleotide, and may be interspersed either individually amongnucleotides or in one or more contiguous groups within SEQ ID NO: 1.

Similarly, a polypeptide having an amino acid sequence of at least 98%“identity” to a sequence contained in SEQ ID NO: 2 means that the aminoacid sequence of the polypeptide is identical to a sequence contained inSEQ ID NO: 2 except that the polypeptide sequence may include up to 2amino acid alterations per each 100 amino acids of the total length ofSEQ ID NO: 2. In other words, to obtain a polypeptide having an aminoacid sequence at least 98% identical to SEQ ID NO: 2, up to 2% of theamino acid residues in the sequence contained in SEQ ID NO: 2 can bedeleted, inserted, or substituted with other amino acid residues. Thesechanges may occur anywhere throughout the polypeptide, and may beinterspersed either individually among residues or in one or morecontiguous groups within SEQ ID NO: 2.

“A polypeptide having biological activity” refers to a polypeptideexhibiting activity similar, but not necessarily identical, to anactivity of a polypeptide of the present invention (e.g. cholesteroltransport activity), as measured in a particular biological assay, withor without dose dependency. In the case where dose dependency doesexist, it need not be identical to that of the polypeptide, but rathersubstantially similar to the dose-dependence in a given activity ascompared to the polypeptide of the present invention (i.e., thecandidate polypeptide will exhibit greater activity or not more thanabout 25-fold less and, preferably, not more than about tenfold lessactivity, and most preferably, not more than about three-fold lessactivity relative to the polypeptide of the present invention.).

“Polypeptide variant” refers to a polypeptide differing from the ABC1polypeptide of the present invention, but retaining essential propertiesthereof. Generally, variants are overall closely similar, and, in manyregions, identical to the polypeptide comprising SEQ ID NO: 2.Preferably, the polypeptide variant retains its biological activity,i.e., cholesterol transport activity. Variants include, but are notlimited to, splice variants and allelic variants, as well as addition,deletion, and substitution variants.

Likewise, “polynucleotide variant” refers to a polynucleotide differingfrom the polynucleotide of the present invention, but retainingessential properties thereof. The variants may contain alterations inthe coding regions, non-coding regions, or both. Thus, for example, anABC1 polynucleotide variant has a nucleotide sequence that differs fromthat of SEQ ID NO: 1, but encodes a polypeptide that has cholesteroltransport activity. Also, for example, a polynucleotide variant has anucleotide sequence that differs from that of SEQ ID NO: 3, but retainspromoter activity. Especially preferred are polynucleotide variantscontaining alterations that produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedpolypeptide. Nucleotide variants produced by silent substitutions due tothe degeneracy of the genetic code are preferred. Moreover, variants inwhich 10-20, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, oradded in any combination are also preferred. Polynucleotide variants canbe produced for a variety of reasons, e.g., to optimize codon expressionfor a particular host (e.g. changing codons in the human mRNA to thosepreferred by a bacterial host such as E. coli).

“Allelic variants” are naturally-occurring variants that refer to one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985).) These allelic variants can vary at either thepolynucleotide and/or polypeptide level. Alternatively, non-naturallyoccurring variants may be produced by mutagenesis techniques or bydirect synthesis.

The term “conservative amino acid substitution” refers to a substitutionof a native amino acid residue with a normative residue such that thereis little or no effect on the polarity or charge of the amino acidresidue at that position. For example, a conservative substitutionresults from the replacement of a non-polar residue in a polypeptidewith any other non-polar residue. Another example of a conservativesubstitution is the replacement of an acidic residue with another acidicresidue. Conservative substitutions are expected to produce ABC1polypeptides having functional and chemical characteristics similar tothose of the naturally-occurring ABC1 polypeptide.

The term “ortholog” refers to a polypeptide that corresponds to apolypeptide identified from a different species. For example, mouse andhuman ABC1 polypeptides are considered orthologs.

The term “vector” is used to refer to any molecule (e.g. nucleic acid,plasmid, or virus) used to transfer coding informationto a host cell.

The term “expression vector” refers to a vector that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control the expression of inserted heterologous nucleicacid sequences. Expression includes, but is not limited to, processessuch as transcription, translation, and RNA splicing, if introns arepresent.

As used herein, the term “transcriptional regulatory region” or“expression modulating portion” refers to any region of the gene,including, but not limited to, promoters, enhancers, and repressors.

As used herein, the term “promoter” refers to an untranscribed sequencelocated upstream (i.e., 5′) to the start codon of a structural gene(generally within about 100 to 1000 bp) that controls the transcriptionof the structural gene.

As used herein the term “enhancers” refers to cis-acting elements ofDNA, usually about 10-300 bp in length, that act on the promoter toincrease transcription. Enhancers are relatively orientation andposition independent. They have been found 5′ and 3′ to thetranscription unit.

“Host cell” is a cell that has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence. The term includes the progeny of the parent cell, whether ornot the progeny is identical in morphology or in genetic make-up to theoriginal parent, so long as the selected gene is present.

The term “operatively linked” is used herein to refer to an arrangementof flanking sequences wherein the flanking sequences so described areconfigured or assembled so as to perform their usual function. Thus, aflanking sequence operably linked to a coding sequence may be capable ofeffecting the replication, transcription and/or translation of thecoding sequence. For example, a coding sequence is operably linked to apromoter when the promoter is capable of directing transcription of thatcoding sequence. A flanking sequence need not be contiguous with thecoding sequence, so long as it functions correctly. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween a promoter sequence and the coding sequence and the promotersequence can still be considered “operably linked” to the codingsequence.

The term “transfection” is used to refer to the uptake of foreign orexogenous DNA by a cell, and a cell has been “transfected” when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art and are disclosedherein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook etal., Molecular Cloning, A Laboratory Manual (Cold Spring HarborLaboratories, 1989); Davis et al., Basic Methods in Molecular Biology(Elsevier, 1986); and Chu et al., 1981, Gene 13:197. Such techniques canbe used to introduce one or more exogenous DNA moieties into suitablehost cells.

ABC1 Polypeptodes

The present invention relates to novel human ABC1 polypeptides. In oneembodiment, the ABC1 polypeptide comprises the amino acid sequence shownin SEQ ID NO: 2. In contrast to the human ABC1 protein reported byothers, the ABC1 polypeptide shown in SEQ ID NO: 2 has an additional 60amino acids at the amino terminus, revealing an ABC1 protein of 2261amino acids rather than 2201 amino acids (see Langmann et al. inBiochem. Biophys. Res. Comm., 257, 29-33 (1999)). In addition, the ABC1polypeptide shown in SEQ ID NO: 2 differs from other reported sequencesat several amino acid residues. Specifically, the present ABC1polypeptide shown in SEQ ID NO: 2 has a K for R at residue 159, I for Vat 765, M for 1 at 823, I for T at 1495, L for P at 1588, K for R at1914, and L for P at 2108. To remain consistent with published notation,the above amino acid numbers are those of Lawn et al., J. Clin. Invest.,104: R25-31 (1999), rather than those of SEQ ID NO: 2. As discussed infurther detail below, the sequence difference likely arises from thefact that the first ABC1 cDNA was cloned from mouse using a PCR-basedstrategy and the subsequently reported human ABC1 cDNA sequences werepredicted from the sequence of the mouse protein. The ABC1 protein hasan approximate molecular weight of 240 kD as determined by SDS-PAGE.

The present invention also relates to ABC1 polypeptides comprising aminoacid sequences which preferably have at least 98% identity over theirentire length to the amino acid sequence of SEQ ID NO: 2. Morepreferably, the polypeptide has at least 99% identity over its entirelength to the amino acid sequence of SEQ ID NO: 2. Most preferably, thepolypeptide has 100% identity over its entire length to the amino acidsequence of SEQ ID NO: 2. As defined previously, the term “identity”refers to the degree of sequence relatedness between polypeptidesequences, which is further defined below.

Such related ABC1 polypeptides include substitution, deletion, andinsertion variants, as well as allelic variants, splice variants,fragments, derivatives, and orthologs. Preferred polypeptides andpolypeptides fragments include those polypeptides and fragments thatpossess the biological activity of ABC1. In particular, thosepolypeptides and fragments that mediate reverse cholesterol transportare preferred. Also preferred are polypeptides and fragments that haveimproved reverse cholesterol transport activity.

Substitution, deletion, and insertion variants refer to ABC1polypeptides comprising amino acid sequences that contain one or moreamino acid sequence substitutions, deletions, and/or additions ascompared to the ABC1 amino acid sequence set forth in SEQ ID NO: 2. Inpreferred embodiments, the variants have from about 1 to 5, or fromabout 1 to 10, or from about 1 to 20, or from about 1 to 40, or fromabout 1 to 65 amino acid substitutions, additions, and/or deletions. Forexample, the variants can have an addition of one or more amino acidresidues anywhere in the polypeptide as well as at the carboxyl terminusand/or at the amino terminus, as long as the variant retains biologicalfunction. Also, for example, one or more amino acids can be deleted fromany region of the polypeptide, including the carboxyl terminus and/oramino terminus, without substantial loss of biological function (Ron etal., J. Biol. Chem., 268: 2984-2988 (1993); Dobeli et al., J.Biotechnology, 7: 199-216 (1988)). The amino acid substitution(s) can beconservative, non-conservative, or any combination thereof, as long asthe ABC1 variant retains its biological activity. In addition, thesubstitution(s) can be with non-conserved amino acid residues, where thesubstituted residues may or may not be encoded by the genetic code, andwith amino acid residues having a substituent group.

Suitable variants of ABC1 polypeptides can be determined usingwell-known techniques. For example, suitable ABC1 variants can bedetermined by identifying regions of the ABC1 molecule that may bechanged without destroying biological activity. Also, as realized in theart, even regions that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure. Amino acid residues that can bechanged without destroying biological activity can be determined byidentifying regions of the ABC1 polypeptide that are not important foractivity (Bowie et al., Science, 247: 1306-1310 (1990)). For example,ABC1 polypeptides from various species can be compared to determine theamino acid residues and regions of ABC1 molecules that are conservedacross species. The conserved amino acid residues are likely importantfor biological function and/or structure. In contrast, changes inregions of the ABC1 molecule that are not conserved across species andare thus tolerated by natural selection would be less likely toadversely affect biological activity and/or structure. Accordingly, ABC1polypeptides with additions, deletions, or substitutions in thenon-conserved regions are likely suitable variants. Even in relativelyconserved regions, chemically similar amino acids may be substituted forthe naturally occurring residues while retaining activity.

In addition, suitable ABC1 variants can be identified usingstructure-function studies to determine residues in other members of theATP-binding cassette protein family, such as ABCR and ABC-C, that areimportant for activity or structure. Such studies allow the predictionof important amino acid residues in an ABC1 variant that correspond toamino acid residues that are important for activity or structure inother ATP-binding cassette proteins. For example, based onstructure-function studies of other ATP-binding cassette proteins,important amino acid residues in ABC1 are likely found in regionsassociated with nucleotide binding and sterol transport. Suitablevariants include, for example, polypeptides having chemically similaramino acid substitutions for such predicted important amino acidresidues of the ABC1 polypeptide.

Suitable ABC1 variants can also be determined using genetic engineeringtechniques to introduce amino acid changes at specific positions inorder to identify regions critical for polypeptide function. Amino acidchanges can be made using, for example, site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science, 244: 1081-1085(1989)). The resultant ABC1 variants can then be tested for biologicalactivity using, for example, any one of the cholesterol efflux assaysdescribed herein. Variants having a particular amino acid residuesubstitution that results in destroyed cholesterol efflux activity wouldnot be considered a suitable ABC1 variant.

Additional methods for identifying suitable variants are known in theart. Furthermore, one skilled in the art would realize amino acidchanges that are likely to be permissive at certain amino acid positionsin the protein (Bowie et al., supra (1990)). For example, it isgenerally known that the most buried or interior (within the tertiarystructure of the protein) amino acid residues require nonpolar sidechains, whereas fewer features of surface or exterior side chains aregenerally conserved. Moreover, it is known that tolerated conservativeamino acid substitutions involve replacement of the aliphatic orhydrophobic amino acids Ala, Val, Leu and Ile, replacement of thehydroxyl residues Ser and Thr, replacement of the acidic residues Aspand Glu, replacement of the amide residues Asn and Gln, replacement ofthe basic residues Lys, Arg, and His, replacement of the aromaticresidues Phe, Tyr, and Trp, and replacement of the small-sized aminoacids Ala, Ser, Thr, Met, and Gly.

The ABC1 variants can be naturally-occurring or artificiallyconstructed. Examples of naturally-occurring variants are allelicvariants and splice variants. Allelic variants refer to one of severalalternate forms of a gene occupying a given locus on a chromosome of anorganism or population of organisms (Lewin, B., ed., Genes II, JohnWiley & Sons, New York (1985)). Allelic variants can vary at either thepolynucleotide and/or polypeptide level. Splice variants refer to anucleic acid molecule, usually RNA, which is generated by alternativeprocessing of intron sequences in an RNA transcript, and thecorresponding polypeptide. Alternatively, the ABC1 variants can beartificially constructed. For example, ABC1 variants can be constructedusing the technique of site-directed mutagenesis. Also, for example,ABC1 variants can be prepared from the corresponding nucleic acidmolecules encoding said variants, which have a DNA sequence that variesas described from the wild-type DNA sequence as set forth in SEQ ID NO:1.

Polypeptide fragments refer to polypeptides which comprise less than thefull length amino acid sequence of ABC1 set forth in SEQ ID NO: 2.Preferred polypeptides fragments include those fragments that possessthe biological activity of ABC1. In particular, those fragments thatmediate reverse cholesterol transport or have improved reversecholesterol transport activity are preferred. ABC1 fragments can haveone or more amino acids deleted from any region of the polypeptide,including the carboxyl terminus and/or amino terminus, as long asbiological function is maintained. The ABC1 polypeptide fragments canoccur naturally, such through alternative splicing or in vivo proteaseactivity, or can be artificially constructed using well-known methods.

The invention also relates to ABC1 polypeptide derivatives, which referto ABC1 polypeptides, variants, or fragments, as defined herein, thathave been chemically modified. The derivatives are modified in a mannerthat is different from naturally-occurring ABC1 polypeptides, either intype or location of the molecules attached to the polypeptide.Derivatives may further include polypeptides formed by the deletion ofone or more chemical groups which are naturally attached to the ABC1polypeptides. In addition, the ABC1 polypeptide comprising the aminoacid sequence of SEQ ID NO: 2, as well as the above-described ABC1variants and fragments, may be fused to a homologous polypeptide to forma homodimer or to a heterologous polypeptide to form a heterodimer.

Another aspect of the present invention relates to mutant ABC1polypeptides and fragments thereof, corresponding to polypeptidesisolated from Tangier patients. In one preferred embodiment, the ABC1polypeptide comprises SEQ ID NO: 8. The protein was isolated from aTangier patient (TD1) and sequenced as described in Examples 1 and 5.The amino acid sequence set forth in SEQ ID NO: 8 is similar to thewild-type sequence with the exception of a glutamine to arginine residuesubstitution at position 537 (the residue number is that of Lawn et al.,J. Clin. Invest., 104: r25-31 (1999), corresponding to position 597 ofSEQ ID NO: 8). The location of this residue is within the amino-terminalhydrophilic domain, near the first predicted transmembrane domain. Thesubstitution alters the charge of the amino acid in this region of theprotein, resulting in an ABC1 protein that has significantly decreasedcholesterol efflux activity, as shown in FIG. 1.

In another preferred embodiment, the mutant ABC1 polypeptide comprisesSEQ ID NO: 10. The protein corresponds to a polypeptide isolated from aTangier patient (TD2) and sequenced as described in Examples 1 and 5.The amino acid sequence set forth in SEQ ID NO: 10 is similar to thewild-type sequence with the exception of an arginine to tryptophansubstitution at residue 527 (the residue number is that of Lawn et al.,supra (1999), corresponding to position 587 of SEQ ID NO: 10). Like theTD1 polypeptide, this substitution alters the charge of the amino acidresidue in the amino-terminal hydrophilic domain of the ABC1 protein.The resultant mutant ABC1 protein also has significantly decreasedcholesterol efflux activity, as shown in FIG. 1.

ABC1 Polynucleotides

Another aspect of the present invention relates to isolatedpolynucleotides that encode the novel ABC1 polypeptides and variantsthereof. The present invention provides, for example, isolatedpolynucleotides encoding the full-length ABC1 polypeptide,polynucleotides containing the full-length cDNA of wild-type ABC1,polynucleotides containing the entire length of the coding sequence ofwild-type ABC1, and polynucleotides containing non-coding 5′ and 3′sequences of ABC1, as well as polynucleotides of related ABC1 variants.The present invention also provides isolated polynucleotides that encodemutant ABC1 polypeptides, such as those of Tangier patients.

In one preferred embodiment, the isolated polynucleotide comprises anucleotide sequence encoding the polypeptide comprising SEQ ID NO: 2.Importantly, in contrast to the published sequence of Langmann et al.which codes for a protein of 2201 amino acids based on a predicted startmethionine found in exon 3 (Langmann et al., Biochem. Biophys. Res.Comm., 257: 29-33 (1999) (GenBank Accession No. AJO12376), the presentlyclaimed nucleotide sequence contains 50 exons and codes for a protein of2261 amino acids (see FIG. 4). The corresponding nucleotide sequence ofthe present invention contains a coding sequence that includes anadditional 180 nucleotides at the 5′ end corresponding to the following60 amino-terminal amino acids:

MACWPQLRLLLWKNLTFRRRQTCQLLLEVAWPLFIFLILISVRLSYPPYEQHECHFPNKA (SEQ ID NO:58). Given that there is an in-frame stop codon 6 to 9 nucleotidesupstream from this location, the newly predicted start site is the firstmethionine codon that could produce a continuous open reading frame.Alignment of this new ABC1 cDNA sequence with related ABC transportersequences ABCR and ABC-C (also known as ABC3) which also contain openreading frames for the 60 additional amino acids, indicates a highdegree of similarity, implying that the homologous ABC transporterproteins begin with sequences related to the amino terminal extensionsequence proposed for human ABC1. It is likely that the earlierpublished start site of the human ABC1 was predicted from the publishedmouse ABC1 cDNA sequence (Luciani et al., Genomics, 21150-159 (1994);GenBank Accession no.: X75926) which contains an extra nucleotide “n” inthe extension region such that the newly disclosed methionine is notin-frame. However, if the “n” nucleotide in the mouse sequence isignored, the mouse and human sequences of the extension region areidentical. In light of these results, it is likely that the full lengthhuman ABC1 protein contains 2261 amino acids rather than 2201 aminoacids, as previously suggested by Langmann et al. and others.Accordingly, Langmann et al. do not present the full open reading frameof human ABC1.

In another preferred embodiment, the isolated polynucleotide comprisesthe full-length ABC1 cDNA, including at least a portion of eithernon-coding 5′ and 3′ sequences. Preferably, the polynucleotide comprisesthe nucleotide sequence shown in SEQ ID NO: 1. The 10.4 kb human ABC1cDNA sequence shown in SEQ ID NO: 1 contains an open reading frame of6783 nucleotides plus 5′ and 3′ untranslated regions. There is a startcodon at position 291 and a stop codon at position 7074. The presentABC1 cDNA shown in SEQ ID NO: 1 differs from the published ABC1 cDNA(GenBank Accession No. AJO12376) in several respects. First, the presentABC1 cDNA includes an additional 350 nucleotides at the 5′ end and anadditional 3136 nucleotides at the 3′ end (not including the poly(A)tail). The present ABC1 sequence also differs from the published ABC1cDNA by the substitution of 10 nucleotides in the coding region. Of theten differences, seven nucleotide differences predict amino acidchanges. To remain consistent with published notation, the followingnucleotide and amino acid numbers are those of Lawn et al., J. Clin.Invest., 104: R25-31 (1999) and GenBank Accession No. AJO12376, ratherthan those of SEQ ID NO: 1. The nucleotide and amino acid changes are asfollows: (1) A for G at nucleotide 414; (2) A for G at nucleotide 596 (Kfor R at amino acid 159); (3) T for C at nucleotide 705; (4) A for C atnucleotide 1980; (5) A for G at 2413 (I for V at amino acid 765); (6) Gfor A at 2589 (M for I at amino acid 823); (7) T for C at 4604 (I for Tat amino acid 1495); (8) T for C at 4883 (L for P at amino acid 1588);(9) A for G at 5861 (K for R at amino acid 1914); and (10) T for C at6443 (L for P at amino acid 2108). Five of the amino acid changes areconservative amino acid changes and may represent polymorphisms orsequence errors. In two instances, the present sequence predictsimportant amino acid differences from the GenBank sequence. Thedifferences result in a leucine rather than a proline at residue 1588and at residue 2108. Interestingly, at both positions, the predictedleucine was also found in each of the three TD samples analyzed as wellas the highly conserved mouse ABC1 protein sequence.

The present invention also relates to ABC1 polynucleotides comprisingnucleotide sequences that preferably have at least 80% identity overtheir entire length to the polynucleotide comprising SEQ ID NO: 1. Morepreferably, the polynucleotide has at least 90% identity over its entirelength to the polynucleotide comprising SEQ ID NO: 1. Even morepreferably, the polynucleotide has at least 95% identity over its entirelength to the polynucleotide comprising SEQ ID NO: 1. Most preferablythe polynucleotide has 100% identity over its entire length to thepolynucleotide comprising SEQ ID NO: 1. Such related ABC1polynucleotides include substitution, deletion, and insertion variants,as well as allelic variants, splice variants, fragments, derivatives,and orthologs, wherein one or more nucleotides have been substituted,deleted, inserted, or derivatized. Preferred polynucleotides includethose polynucleotides that encode ABC1 polypeptides possessingbiological activity, such as cholesterol efflux activity.

In another preferred embodiment, the isolated polynucleotide comprisesthe entire coding sequence of ABC1. In a particularly preferredembodiment, the polynucleotide comprises the sequence shown asnucleotides 291-7074 of SEQ ID NO: 1. This isolated polynucleotidecontains an ABC1 open reading frame of 6783 nucleotides and encodes apolypeptide of 2261 amino acids, as described above.

In yet another preferred embodiment, the isolated polynucleotidecomprises a nucleotide sequence that encodes an ABC1 variantpolypeptide. In particular, the isolated polynucleotide comprises anucleotide sequence that encodes a polypeptide comprising an amino acidsequence which is at least 98% identical to the amino acid sequence ofSEQ ID NO: 2. Also preferred is an isolated polynucleotide comprising anucleotide sequence that encodes a polypeptide comprising an amino acidsequence which is at least 99% identical to the amino acid sequence ofSEQ ID NO: 2. Accordingly, the invention includes those polynucleotidesthat encode the above-described ABC1 polypeptides, including thedescribed substitution, deletion, and insertion variants, as well asABC1 allelic variants, splice variants, fragments, derivatives, fusionpolypeptides, and orthologs. Preferred polynucleotides are thosepolynucleotides that encode polypeptides that possess the biologicalactivity of ABC1. In particular, those polynucleotides that encodepolypeptides that mediate reverse cholesterol transport are preferred.Also preferred are polynucleotides that enocode polypeptides that haveimproved reverse cholesterol transport activity.

Yet another aspect of the invention relates to isolated polynucleotidesthat encode mutant ABC1 polypeptides from Tangier patients. In onepreferred embodiment, the polynucleotide encodes the polypeptide of SEQID NO: 8, which polypeptide is isolated from patient TD1 and isdescribed above. In another preferred embodiment, the polynucleotidecomprises the nucleotide sequence set forth in SEQ ID NO: 7. Thenucleotide sequence set forth in SEQ ID NO: 7 contains the full openreading frame, as well as 5′ and 3′ flanking sequences. The open readingframe encodes a polypeptide of 2261 amino acids, containing, among othersubstitutions, a nucleotide substitution that results in an A to Gsubstitution at position 537 (using the numbering of Lawn et al., supra(1999)).

In another preferred embodiment relating to polynucleotides that encodemutant ABC1 polypeptides, the polynucleotide encodes the polypeptide ofSEQ ID NO: 10, which polypeptide is isolated from Tangier patient TD2and is also described above. In yet another preferred embodiment, thepolynucleotide comprises the nucleotide sequence set forth in SEQ ID NO:9. The nucleotide sequence set forth in SEQ ID NO: 9 contains the fullopen reading frame, as well as 5′ and 3′ flanking sequences. The openreading frame encodes a polypeptide of 2261 amino acids, containing,among other substitutions, a polynucleotide substitution that results inan Arg to Tryp substitution at residue 527 (using the numbering of Lawnet al., supra (1999)).

Another aspect of the present invention relates to isolatedpolynucleotides that comprise the non-coding 5′ flanking and 3′ flankingregions of ABC1. In one embodiment, the isolated polynucleotidecomprises the non-coding 5′ flanking region of ABC1. Preferably, the 5′flanking region contains, but is not limited to, the ABC1 promoterregion. Thus, in a preferred embodiment, the polynucleotide comprisesthe sequence shown in SEQ ID NO: 3. As demonstrated by heterologousreporter assays, discussed in Example 15, the polynucleotide set forthin SEQ ID NO: 3 contains the transcriptional regulatory region of theABC1 gene. As shown in FIG. 13, the polynucleotide set forth in SEQ IDNO: 3 is a 1643 b.p. non-coding sequence that contains severaltranscription regulatory elements, including a TATA box at positions1522, 1435, and 1383, as well as transcription factor binding sites,including several putative SPI sites, and several nuclear receptor halfsites. In addition, an identified sterol response element is found atposition 1483-1500. Further heterologous reporter assays described inExample 18 revealed that several discrete portions of SEQ ID NO: 3retained promoter activity. Accordingly, in another preferredembodiment, the polynucleotide comprises nucleotides 1-1532, 1080-1643,1181-1643, 1292-1643, or 1394-1643 of SEQ ID NO: 3. In an especiallypreferred embodiment, the polynucleotide comprises nucleotides 1394-1532of SEQ ID NO: 3, which sequence has been shown to have ABC1 promoteractivity (see Example 18). In yet another preferred embodiment, thepolynucleotide comprises nucleotides 1480-1510 of SEQ ID NO: 3, which isshown to regulate the ABC1 transcriptional response to LXR ligands.

The 5′ flanking polynucleotide also comprises a nucleotide sequence thathybridizes, under stringent conditions, to the nucleotide sequence setforth in SEQ ID NO: 3, wherein the nucleotide sequence has ABC1 promoteractivity. In yet another embodiment, the polynucleotide comprises anucleotide sequence that hybridizes, under stringent conditions, to thenucleotide sequence comprising nucleotides 1-1532, 1080-1643, 1181-1643,1292-1643, or 1394-1643 of SEQ ID NO: 3, wherein the nucleotide sequencehas ABC1 promoter activity.

In another embodiment, the isolated polynucleotide comprises the 3′flanking region of ABC1. Several 3′ untranslated regions have beenidentified which may represent alternate sites of polyadenylation of theABC1 transcript. Preferably, the 3′ flanking region contains regulatorysequences. For example, the full length 3′ UTR (SEQ ID NO: 6) contains46 sequences (AA)nCU/UC(AA)n (SEQ ID NO: 59) which have been shown to benecessary for binding of Vigilin. Vigilin, a ubiquitous protein with 14Khomology domains, is the estrogen-inducible vitellogenin mRNA3′-untranslated region binding protein (J. Biol. Chem., 272: 12249-12252(1997)). In addition to binding HDL, Vigilin has been shown to bind tothe 3′ flanking region of mRNAs and to increase the half-life of themRNA transcript (Mol. Cell. Biol., 18:3991-4003 (1998)). Thus, the 3′flanking region could be altered, for example, to increase the bindingof Vigilin, thereby increasing the half-life of the ABC1 mRNA.Preferably, the isolated polynucleotide comprises the sequence shown inSEQ ID NO: 4. Also preferably, the isolated polynucleotide comprises thesequence shown in SEQ ID NO: 5. In another preferred embodiment, theisolated polynucleotide comprises the sequence shown in SEQ ID NO: 6. Inother preferred embodiments, the polynucleotide comprises a sequencethat hybridizes, under stringent conditions, to the nucleotide sequenceset forth in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

The present invention also includes related ABC1 5′ and 3′ flankingpolynucleotides. Accordingly, the invention relates to polynucleotidescomprising nucleotide sequences that have at least 80% identity overtheir entire length to the polynucleotide comprising SEQ ID NO: 3, thepolynucleotide comprising SEQ ID NO: 4, the polynucleotide comprisingSEQ ID NO: 5, the polynucleotide comprising SEQ ID NO: 6, or thepolynucleotide comprising nucleotides 1-1532, 1080-1643, 1181-1643,1292-1643, or 1394-1643 of SEQ ID NO: 3. Preferably, the polynucleotidehas at least 80%, more preferably at least 90%, even more preferably atleast 95%, and most preferably 100% identity over its entire length toany one of the aforementioned flanking polynucleotides. Preferredpolynucleotides include those polynucleotides that possess biologicalactivity, such as transcriptional regulatory activity.

It is understood that the present invention further relates to isolatedpolynucleotides that are complementary to any one of the above-describedpolynucleotide sequences. As used herein, the term “complementary”refers to the hybridization or base pairing between nucleotides, suchas, for example, between the two strands of a double-strandedpolynucleotide. Two single-stranded nucleotide molecules are said to becomplementary when the nucleotides of one strand, optimally aligned withappropriate nucleotide insertions, deletions or substitutions, pair withat least about 80% of the nucleotides of the other strand.

Another aspect of the present invention relates to compositionscomprising the novel ABC1 polynucleotides described above and a suitablecarrier. In one embodiment, the composition comprises a polynucleotideencoding the polypeptide comprising SEQ ID NO: 2, a polynucleotidecomprising SEQ ID NO: 1, a polynucleotide comprising nucleotides291-7074 of SEQ ID NO: 1, or a polynucleotide encoding a polypeptidecomprising an amino acid sequence which is at least 98% identical to theamino acid sequence of SEQ ID NO: 2 and a suitable carrier. In anotherembodiment, the composition comprises a polynucleotide having at least80%, preferably 90%, or more preferably 95% identity over its entirelength to the polynucleotide comprising SEQ ID NO: 1 and a suitablecarrier.

In another embodiment, the composition comprises a polynucleotidecomprising an ABC1 5′ flanking sequence and a suitable carrier.Preferably, the composition comprises a polynucleotide comprising SEQ IDNO: 3 or a polynucleotide comprising nucleotide fragments 1-1532,1080-1643, 1181-1643, 1292-1643, or 1394-1643 of SEQ ID NO: 3 and asuitable carrier. Also preferably, the composition comprises apolynucleotide having at least 80%, 90%, or 95% identity over its entirelength to the polynucleotide comprising any one of the described 5′flanking sequences and a suitable carrier. In yet another embodiment,the composition comprises a polynucleotide comprising an ABC1 3′flanking sequence and a suitable carrier. Preferred compositionscomprise a polynucleotide comprising SEQ ID NO: 4, a polynucleotidecomprising SEQ ID NO: 5, or a polynucleotide comprising SEQ ID NO: 6, aswell as a polynucleotide having at least 80%, preferably 90%, or morepreferably 95% identity over its entire length to any of thesepolynucleotides, and a suitable carrier. Still other compositions of theinvention comprise mutant ABC1 polynucleotides. Preferably, thecomposition comprises a polynucleotide comprising SEQ ID NO: 7 or apolynucleotide comprising SEQ ID NO: 9 and a suitable carrier.

In addition, a composition of the present invention may comprise, in anycombination, two or more of the above-described ABC1 polynucleotides anda suitable carrier. Any suitable aqueous carrier can be used in thecomposition. Preferably, the carrier renders the composition stable at adesired temperature, such as room temperature or storage temperature(i.e. 4° C. to −20° C.), and is of approximately neutral pH. Examples ofsuitable carriers are known to those of skill in the art and includeTris-EDTA buffer and DEPC-H₂O.

ABC1 Vectors and Host Cells

The present invention also relates to recombinant vectors that compriseone or more of the above-described ABC1 polynucleotides, host cells thatare genetically engineered with the vectors comprising ABC1polynucleotides, and the production of ABC1 polypeptides by recombinanttechniques. As mentioned, the invention provides recombinant vectorsthat comprise one or more of the above-described wild-type ABC1polynucleotides. In preferred embodiments, the recombinant vectorcomprises the polynucleotide encoding the polypeptide comprising SEQ IDNO: 2, the polynucleotide comprising SEQ ID NO: 1, and thepolynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1. Inanother preferred embodiment, the recombinant vector comprises thevariant polynucleotide encoding a polypeptide comprising an amino acidsequence which is at least 98% identical to the amino acid sequence ofSEQ ID NO: 2. Also, in another preferred embodiment, the recombinantvector comprises a variant polynucleotide that is at least 80% identicalto a polynucleotide comprising SEQ ID NO: 1. In still another preferredembodiment, the recombinant vector comprises a polynucleotide that iscomplementary to any of these polynucleotides.

In another embodiment, the recombinant vector comprises a polynucleotidecomprising a mutant ABC1 polynucleotide isolated from a Tangier diseasepatient. In a preferred embodiment, the recombinant vector comprises thepolynucleotide comprising SEQ ID NO: 8. In another preferred embodiment,the recombinant vector comprises the polynucleotide comprising SEQ IDNO: 10. The recombinant vectors may also comprise a polynucleotidesequence that is complementary to these sequences.

It is also understood that the recombinant vector may also comprise, inany combination, two or more of the above-described wild-type, variant,or mutant ABC1 polynulceotides.

An isolated ABC1 polynucleotide, such as any of the above-describedwild-type, variant, or mutant polynucleotides, is inserted into a vectorusing well-known ligation and cloning techniques. Cloning techniqueshave been described in several standard laboratory manuals, includingDavis et al., Basic Methods in Molecular Biology (1986); Sambrook etal., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., (Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1989)); Ausubel et al. eds.,Current Protocols in Molecular Biology, (Wiley and Sons (1994));Goeddel, ed., Gene Expression Technology (Methods in Enzymology (1991));Murray, ed., Gene Transfer and Expression Protocols (Human Press,Clifton, N.J.).

Any vector suitable for ABC1 polynucleotide insertion can be used. Thevector is typically selected to be functional in the particular hostcell employed (i.e., the vector is compatible with the host cellmachinery such that amplification and/or expression of the gene canoccur). Preferably, the vector is compatible with bacterial, insect, ormammalian host cells. Also preferably, the vector is an expressionvector (for a review of expression vectors, see Goeddel, D. V. ed.,Methods Enzymol., Academic Press Inc., San Diego, Calif. (1990)). Thevector may be, for example, a phage, plasmid, viral, or retroviralvector. Retroviral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host cells.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” should preferably include one or more of thefollowing nucleotide sequences: a promoter, one or more enhancersequences, an origin of replication, a transcriptional terminationsequence, a complete intron sequence containing a donor and acceptorsplice site, a sequence encoding a leader sequence for polypeptidesecretion, a ribosome binding site, a polyadenylation sequence, apolylinker region for inserting the nucleic acid encoding thepolypeptide to be expressed, and a selectable marker element.

The flanking sequences may be homologous (i.e., from the same speciesand/or strain as the host cell), heterologous (i.e., from a speciesother than the host cell species or strain), hybrid (i.e., a combinationof flanking sequences from more than one source), or synthetic. Also,the flanking sequences may be native sequences which normally functionto regulate ABC1 polypeptide expression. The source of a flankingsequence may be any prokaryotic or eukaryotic organism, any vertebrateor invertebrate organism, or any plant, provided that the flankingsequence is functional in, and can be activated by, the host cellmachinery.

The vector should also preferably include at least one selectable markerfor propagation in a host. A selectable marker is a gene element thatencodes a protein necessary for the survival and growth of a host cellgrown in a selective culture medium. Suitable selection marker genesencode proteins that (a) confer resistance to antibiotics or othertoxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotichost cells; (b) complement auxotrophic deficiencies of the cell; or (c)supply critical nutrients not available from complex media. Preferredselectable markers include zeocin, G418, hygromycin, or neomycinresistance for eukaryotic cell culture and tetracycline, kanamycin, orampicillin resistance for culturing in E. coli and other bacteria.

Other suitable selection genes include those that are used to amplifythe expressed gene. Amplification is the process wherein genes that arein greater demand for the production of a protein critical for growthare reiterated in tandem within the chromosomes of successivegenerations of recombinant cells. Examples of suitable selectablemarkers for mammalian cells include dihydrofolate reductase (DHFR) andthymidine kinase. The mammalian cell transformants are placed underselection pressure wherein only the transformants are uniquely adaptedto survive by virtue of the selection gene present in the vector.Selection pressure is imposed by culturing the transformed cells underconditions in which the concentration of selection agent in the mediumis successively changed, thereby leading to the amplification of boththe selection gene and the ABC1 gene. As a result, increased quantitiesof ABC1 polypeptide are synthesized from the amplified DNA.

The vector should also preferably contain a transcription terminationsequence, which is typically located 3′ of the end of a polypeptidecoding region and serves to terminate transcription. Usually, thetranscription termination sequence in prokaryote cells is a G-C richfragment followed by a poly T sequence. The sequence can be purchased aspart of a commercial vector or synthesized using well-known methods fornucleic acid synthesis.

The vector should also preferably contain a ribosome binding site, whichis usually necessary for translation initiation of mRNA and ischaracterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozaksequence (eukaryotes). The element is typically located 3′ to thepromoter and 5′ to the coding sequence of the ABC1 polypeptide to beexpressed. The Shine-Dalgarno sequence is varied but is typically apolypurine (i.e., having a high A-G content). Many Shine-Dalgarnosequences have been identified, each of which can be readily synthesizedusing well-known methods.

The vector should also preferably contain a promoter that is recognizedby the host organism and operably linked to the encoded polynucleotide.The promoter can be an inducible promoter or a constitutive promoter.Inducible promoters initiate increased levels of transcription from DNAunder their control in response to some change in culture conditions,such as the presence or absence of a nutrient or a change intemperature. In contrast, constitutive promoters initiate continual geneproduct production; consequently there is little or no control over geneexpression. A suitable promoter is operably linked to a polynucleotide,by removing the promoter from the source DNA by restriction enzymedigestion and inserting the desired promoter sequence into the vector.As mentioned, a native promoter can be used to direct amplificationand/or expression of the polynucleotide. Thus, the recombinant vectorcan comprise any one of the above-described wild-type, variant, ormutant ABC1 polynulceotides and an ABC1 promoter, such as that found inSEQ ID NO: 3. The recombinant vector can also comprise, in anycombination, two or more of the above-described wild-type, variant, ormutant ABC1 polynulceotides and an ABC1 promoter.

Preferably, a heterologous promoter is used if it permits greatertranscription and higher yields of ABC1 protein as compared to thenative ABC1 promoter, and if it is compatible with the host cell systemthat has been selected for use. The heterologous promoter can be usedalone or in conjunction with the native ABC1 promoter. Thus, in onepreferred embodiment, the recombinant vector comprises any one of theabove-described wild-type ABC1 polynucleotides and a heterologouspromoter. In another preferred embodiment, the recombinant vectorcomprises any one of the above-described variant ABC1 polynucleotidesand a heterologous promoter. In yet another preferred embodiment, therecombinant vector comprises any one of the above-described mutant ABC1polynucleotides and a heterologous promoter. Preferred embodiments alsoinclude recombinant vectors that contain any combination of two or moreof the above-described wild-type, variant, and mutant ABC1polynucleotides and a heterologous promoter.

Heterologous promoters suitable for use with prokaryotic hosts include,but are not limited to, the beta-lactamase and lactose promoter systems(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.,75:3727-31), alkaline phosphatase, a tryptophan (trp) promoter system,and hybrid promoters such as the tac promoter (Villa-Kamaroff et al.,1978, Proc. Natl. Acad. Sci. U.S.A., 75:3727-31). Their sequences havebeen published, thereby enabling one skilled in the art to ligate themto the desired DNA sequence, using linkers or adapters as needed tosupply any useful restriction sites. Other suitable heterologouspromoters will be known to those skilled in the art.

Suitable heterologous promoters for use with mammalian host cells arealso well known and include, but are not limited to, those obtained fromthe genomes of viruses such as polyoma virus, fowlpox virus, adenovirus(such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and Simian Virus 40(SV40). Other suitable mammalian promoters include heterologousmammalian promoters, for example, heat-shock promoters and the actinpromoter. Additional suitable promoters include, but are not limited to:the SV40 early promoter and late promoter region (Bernoist and Chambon,1981, Nature 290:304-10); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-97);the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:1444-45); and the regulatory sequences of themetallothionine gene (Brinster et al., 1982, Nature 296:39-42).Preferably, the promoter is a cytomegalovirus or SV40 promoter. Thus, inespecially preferred embodiments, the recombinant vector comprises oneof the above-described wild-type ABC1 polynucleotides, one of theabove-described variant ABC1 polynucleotides, or one of theabove-described mutant ABC1 polynucleotides and a cytomegaloviruspromoter. In another especially preferred embodiment, the recombinantvector comprises, in any combination, two or more of the above-describedwild-type, variant, or mutant ABC1 polynulceotides and a cytomegaloviruspromoter.

The vector also preferably contains an enhancer sequence to increase thetranscription of a polynucleotide, such as ABC1. Suitable enhancers forthe activation of eukaryotic promoters include viral enhancers, such asthe SV40, cytomegalovirus early promoter, polyoma, and adenovirusenhancers.

Expression vectors of the invention may be constructed from a startingvector, such as a commercially available vector, which may or may notcontain all of the desired flanking sequences. Where one or more of theflanking sequences described herein are not already present in thevector, they may be individually obtained and ligated into the vector.Methods used for obtaining each of the flanking sequences are well knownto one skilled in the art.

Preferred vectors are those which are compatible with bacterial, insect,and mammalian host cells. Vectors preferred for use in bacteria include,for example, pQE70, pQE60, and pQE-9 (Quiagen, Inc.), pBluescriptvectors, Phagescript vectors, pNH16A, pNH18A, pNH46A (Stratagene CloningSystems, Inc.), ptrc99a, pKK223-3, pDR540, pRIT5 (Pharmacia Biotech,Inc.), and pCEP4 (Invitrogen Corp., Carlsbad, Calif.). Preferredeukaryotic vectors include, but are not limited to, pWLNEO, pSV2CAT,pOG44, pXTI and pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL(Pharmacia), and pGL3 (Promega, Madison, Wis.). Other suitable vectorswill be readily apparent to the skilled artisan.

In an especially preferred embodiment, the recombinant vector comprisespCEPhABC1, which is described in Example 4 and shown in FIG. 3. Therecombinant vector pCEPhABC1 comprises the plasmid pCEP4 (Invitrogen),an expression vector containing the cytomegalovirus promoter andenhancer. The vector pCEPhABC1 further comprises an ABC1 polynucleotideoperatively linked to the heterologous cytomegalovirus promoter. TheABC1 polynucleotide comprises SEQ ID NO: 1, which contains thefull-length ABC1 cDNA, including non-coding 5′ flanking (i.e., nativeABC1 promoter) and 3′ flanking sequences.

In addition, the present invention provides recombinant vectorscomprising ABC1 flanking sequence polynucleotides. In one embodiment,the recombinant vector comprises a polynucleotide comprising a ABC1 5′flanking sequence that preferably contains promoter activity. Thus, in apreferred embodiment, the recombinant vector comprises thepolynucleotide comprising SEQ ID NO: 3. In another preferred embodiment,the recombinant vector comprises the polynucleotide comprisingnucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1643 of SEQID NO: 3. In an especially preferred embodiment, the polynucleotidecomprises nucleotides 1394-1532 of SEQ ID NO: 3. Also, in anotherembodiment, the recombinant vector comprises a polynucleotide thathybridizes, under stringent conditions, to the polynucleotide set forthin SEQ ID NO: 3 or the polynucleotide comprising nucleotides 1-1532,1080-1643, 1181-1643, 1292-1643, or 1394-1643 of SEQ ID NO: 3. In yetanother embodiment, the polynucleotide comprises a nucleotide sequencethat has at least 80%, more preferably at least 90%, and even morepreferably at least 95% identity over its entire length to thepolynucleotide comprising SEQ ID NO: 3 or the polynucleotide comprisingnucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1643 of SEQID NO: 3. The recombinant vector also comprises a polynucleotide that iscomplementary to any of the above-described 5′ flanking sequences.

In another embodiment, the recombinant vector comprises a polynucleotidecomprising a 3′ flanking sequence of ABC1. In a preferred embodiment,the recombinant vector comprises the polynucleotide comprising SEQ IDNO: 4. In another preferred embodiment, the recombinant vector comprisesthe polynucleotide comprising SEQ ID NO: 5. In an equally preferredembodiment, the recombinant vector comprises the polynucleotidecomprising SEQ ID NO: 6. Also, in another embodiment, the recombinantvector comprises a polynucleotide that hybridizes, under stringentconditions, to the polynucleotide set forth in SEQ ID NO: 4, SEQ ID NO:5, or SEQ ID NO: 6. In yet another embodiment, the polynucleotidecomprises a nucleotide sequence that has at least 80%, more preferablyat least 90%, and even more preferably at least 95% identity over itsentire length to the polynucleotide comprising SEQ ID NO: 4, SEQ ID NO:5, or SEQ ID NO: 6. The recombinant vector also comprises apolynucleotide that is complementary to any of the above-described 3′flanking sequences.

An isolated ABC1 flanking sequence polynucleotide, such as any of theabove-described 5′ or 3′ flanking sequence polynucleotides, is insertedinto a vector using well-known ligation and cloning techniques. Any ofthe previously described vectors can be used. Preferably, the vector iscompatible with bacterial, insect, or mammalian host cells. Alsopreferably, the vector is an expression vector. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector.

In addition to the ABC1 flanking sequence, the vector may contain one ormore of the following flanking nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Any ofthe previously described flanking nucleotide sequences are suitable. Theflanking sequences may be homologous, heterologous, hybrid, orsynthetic. Also, the flanking sequences may be native sequences whichnormally function to regulate ABC1 polypeptide expression. The source ofa flanking sequence may be any prokaryotic or eukaryotic organism, anyvertebrate or invertebrate organism, or any plant, provided that theflanking sequence is functional in, and can be activated by, the hostcell machinery.

Preferred vectors are those which are compatible with bacterial, insect,and mammalian host cells. Suitable vectors have been previouslydescribed and include pQE70, pQE60, pQE9, pBluescript vectors,Phagescript vectors, pNH16A, pNH18A, pNH46A, ptrc99a, pKK223-3, pDR540,pRIT5, and pCEP4 for use in bacteria and pWLNEO, pSV2CAT, pOG44, pXT1,pSG, pSVK3, pBPV, pMSG, pSVL, and pGL3 for use in eukaryotic cells.

In one particularly preferred embodiment, the recombinant vectorcomprises a polynucleotide comprising the 5′ flanking region of the ABC1gene and further comprises at least one polynucleotide encoding aheterologous polypeptide. The heterologous polynucleotide is operativelylinked to the ABC1 5′ flanking sequence. The ABC1 5′ flanking sequencepreferably contains the ABC1 promoter. Thus, preferably, the 5′ flankingsequence comprises the sequence set forth in SEQ ID NO: 3. Equallypreferably, the 5′ flanking sequence comprises nucleotides 1-1532,1080-1643, 1181-1643, 1292-1643, or 1394-1643 of SEQ ID NO: 3. Theheterologous polynucleotide preferably encodes a polypeptide that is acomplete protein or a biologically active fragment of a protein. Thevector may also contain more than one heterologous polynucleotide. Morepreferably, the heterologous polynucleotide encodes a reporter protein.In such case, the recombinant vector preferably does not contain anyadditional promoter sequences. Examples of suitable reporter proteinsinclude luciferase, β-galactosidase, chloramphenicol acetyl transferase,and green fluorescent protein. Preferably, the reporter polypeptide isluciferase. Thus, in one especially preferred embodiment, therecombinant vector comprises the 5′ flanking sequence set forth in SEQID NO: 3 and a luciferase reporter polynucleotide. In other equallypreferred embodiments, the recombinant vector comprises a polynucleotidecomprising nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, or1394-1643 of SEQ ID NO: 3 and a luciferase reporter polynucleotide.

Expression vectors comprising the ABC1 5′ flanking sequence can beconstructed from a starting vector, such as a commercially availablevector, which contains a reporter polynucleotide. Examples of suitableexpression vectors include pGL3-Basic, which contains a luciferasereporter gene (Promega, Madison, Wis.) and pβGal-Basic (Clontech, PaloAlto, Calif.). A preferred vector is the pGL3-Basic luciferase reportervector, which is promoterless. A 5′ flanking sequence containing theABC1 promoter, for example SEQ ID NO: 3, can be ligated into one of theabove expression vectors using well-known methods, including the methodsdescribed herein (see Example 15). Thus, in an especially preferredembodiment, the recombinant vector is pAPR1, a reporter gene constructcomprising SEQ ID NO: 3 and a luciferase reporter gene in a pGL3 vector(see FIG. 11).

The present invention also relates to host cells comprising any one ofthe above-described recombinant vectors. After the vector has beenconstructed, the completed vector can be inserted into a suitable hostcell for amplification and/or polypeptide expression. Host cells may beprokaryotic host cells (such as E. coli) or eukaryotic host cells (suchas a yeast cell, an insect cell, or a vertebrate cell). The host cell,when cultured under appropriate conditions, synthesizes an ABC1polypeptide or, alternatively, a reporter polypeptide, which can besubsequently measured. The host cell can be a mammalian host cell, suchas a primate cell line or a rodent cell line, including transformed celllines. Normal diploid cells, cell strains derived from in vitro cultureof primary tissue, as well as primary explants, are also suitable.Candidate host cells may be genotypically deficient in the selectiongene, or may contain a dominantly acting selection gene.

A number of suitable host cells are known in the art and many areavailable from the American Type Culture Collection (ATCC), Manassas,Va. Suitable mammalian host cells include, but are not limited to,chinese hamster ovary cells (CHO), CO DHFR-cells (Urlaub et al., 1980,Proc. Natl. Acad. Sci., 97:4216-20), human embryonic kidney (HEK) 293 or293T cells, or 3T3 cells, monkey COS-1 and COS-7 cell lines, and theCV-1 cell line. Other suitable mammalian cell lines include but are notlimited to, mouse neuroblastoma N2A cells, HeLa cells, mouse 1-929cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HAKhamster cell lines, Thp-1, HepG2, and mouse RAW cell lines. Each ofthese cell lines is known by and available to those skilled in the artof protein expression. A preferred host cell is the mouse monocytic cellline RAW 264.7, which use is described in Example 8.

Suitable bacterial host cells include various strains of E. coli (e.g.,HB101, DH50, DH11, and MC 1061), which are well-known as host cells inthe field of biotechnology. Various strains of B. subtilis, Pseudomonasspp., other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method. Also, many strains of yeast cells are alsoavailable as host cells for the expression of ABC1 polypeptides.Preferred yeast cells include, for example, Saccharomyces cerivisae andPichia pastoris. Additionally, insect cell systems may be suitable hostcells. Insect cell systems are described, for example, in Kitts et al.,1993, Biotechniques, 14:810-17; Lucklow, 1993, Curr. Opin. Biotechnol.4:564-72; and Lucklow et al., 1993, J. Virol., 67:4566-79. Preferredinsect cells are Sf-9 and Hi5 (Invitrogen).

The present invention also provides a method for expressing an ABC1protein in a mammalian host cell comprising the steps of: (a)transfecting the mammalian host cell with a recombinant expressionvector comprising a polynucleotide encoding ABC1 in an amount sufficientto produce a detectable level of ABC1 protein; and (b) purifying theproduced ABC1 protein. In one preferred embodiment, the recombinantexpression vector comprises a polynucleotide encoding the polypeptidecomprising SEQ ID NO: 2. In another preferred embodiment, thepolynucleotide comprising SEQ ID NO: 1. In yet another preferredembodiment, the polynucleotide comprising nucleotides 291-7074 of SEQ IDNO: 1. In still another preferred embodiment, the polynucleotideencoding a polypeptide that is at least 98% identical to the polypeptidecomprising SEQ ID NO: 2.

Introduction of the recombinant ABC1 vector into a mammalian host cellcan be effected by methods well-known in the art and described instandard laboratory manuals, such as Sambrook, supra. Preferably, therecombinant vector is introduced into a host cell in a precipitate or ina complex with a charged lipid. Suitable methods for introduction of theABC1 vector include calcium phosphate transfection, DEAE-dextranmediated transfection, cationic lipid-mediated transfection,electroporation, transduction, infection, or other methods known in theart. These methods are described, for example, in Davis et al., BasicMethods in Molecular Biology (1986). If the recombinant ABC1 vector is aviral vector, it may be packaged in vitro using an appropriate packagingcell line and then transduced into host cells.

The ABC1 polypeptide can be recovered and purified from recombinant cellcultures using well-known methods, including ammonium sulfate or ethanolprecipitation, acid, extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatoraphy,and lectin chromatography (see, e.g., Smith and Johnson, Gene 67:31-40(1988)). Preferably, affinity chromotography using anti-ABC1 antibodiesis employed for purification.

In addition, the present invention provides a method for expressing ABC1protein in a mammalian subject comprising the step of administering to amammalian subject a recombinant expression vector comprising apolynucleotide encoding ABC1 in an amount sufficient to express ABC1protein in said mammalian subject. Preferably, the recombinantexpression vector comprises a polynucleotide encoding the polypeptidecomprising SEQ ID NO: 2, the polynucleotide comprising SEQ ID NO: 1, thepolynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1, or thepolynucleotide encoding a polypeptide that is at least 98% identical tothe polypeptide comprising SEQ ID NO: 2. Introduction of the recombinantABC1 vector into a mammalian subject can be effected by methodswell-known in the art and are described in detail herein below.Expression of ABC1 can be measured by obtaining a blood sample from thesubject to whom the recombinant ABC1 vector was administered, separatingthe monocyte population, and measuring the ABC1 gene expression inmacrophage cells. The ABC1 gene expression can be measured using methodswell-known in the art, such as RT-PCR, and methods described herein (seeExamples 9 and 10). The level of ABC1 protein can be measured byobtaining a blood sample from the subject, separating the monocytepopulation and measuring the ABC1 protein in macrophage cells usingwell-known methods, such as immunoprecipitation, described herein atExample 11.

Methods and Compounds for Increasing Cholesterol Efflux

In another aspect of the present invention, a method suitable forincreasing cholesterol efflux from cells in a mammalian subject isprovided. Such method comprises administering to the mammalian subject arecombinant expression vector comprising an ABC1 polynucleotide in anamount sufficient to increase cholesterol efflux from said cells. Therecombinant vector can be any of the above-described vectors containingany of the previously described wild-type or variant ABC1polynucleotides, as long as the encoded ABC1 polypeptide has biologicalactivity (i.e., cholesterol transport activity). Preferably, therecombinant vector comprises the polynucleotide encoding the polypeptidecomprising SEQ ID NO: 2, the polynucleotide comprising SEQ ID NO: 1, thepolynucleotide comprising nucleotides 291-7074 of SEQ ID NO: 1, or thepolynucleotide encoding a polypeptide comprising an amino acid sequencewhich is at least 98% identical to the amino acid sequence of SEQ ID NO:2. Also, preferably, the recombinant vector comprises a variantpolynucleotide that is at least 80%, 90%, or 95% identical to thepolynucleotide comprising SEQ ID NO: 1, as long as the encoded ABC1polypeptide has cholesterol transport activity.

The administration of a recombinant ABC1 expression vector to amammalian subject can be used to express the ABC1 gene in said subjectfor the treatment of cardiovascular disease. Specifically, this methodwould achieve its therapeutic effect by the introduction of the ABC1gene into macrophage cells and other cholesterol-accumulating cellsfound in the arterial lesions of mammals with cardiovascular disease.Expression of the ABC1 polynucleotide in target cells would effectgreater production of the ABC1 protein. The subsequently produced ABC1protein would ameliorate the disease by increasing the efflux ofcholesterol from macrophage and other cholesterol-laden cells found inarterial lesions onto nascent HDL particles. The cellular efflux wouldlead to the overall removal of cholesterol from peripheral sites, suchas the cholesterol-rich core of arterial plaques. A concurrent reductionin the size of these pathological lesions reduces the risk of arterialblockage that leads to heart attacks and angina. This method could alsobe used prophylactically to prevent the accumulation of cholesterol inarterial walls.

A sufficient amount of ABC1 expression vector is the amount of ABC1vector that increases cholesterol efflux from the cells of a mammaliansubject. Such amount can be determined by measuring the cholesterolefflux in the cells of a subject before (control level) and afteradministration of the recombinant ABC1 expression vector at variousdosages and determining the dose that effects an increase in cholesterolefflux compared to control level. The cholesterol efflux can be measuredby obtaining a blood sample from the subject, separating the monocytepopulation, and measuring the amount of cholesterol efflux in asubject's macrophage cells. Any of the assays described herein can beused to measure cholesterol efflux.

In addition, cholesterol efflux can be measured by determining the levelof plasma HDL-cholesterol in a subject before (control) and afteradministration of a recombinant ABC1 expression vector. An observedincrease in HDL-cholesterol in the serum of a subject followingadministration of a recombinant ABC1 expression vector indicates anincrease in cholesterol efflux. HDL-cholesterol levels in serum can bedetermined using methods well-known in the art.

In addition, cholesterol efflux can be measured by measuring the size ofatherosclerotic lesions found in the arterial wall of a subject before(control level) and after administration of the recombinant ABC1expression vector. A reduction in the size of the arterial lesionindicates an increase in cholesterol efflux. Assays for measuringarterial lesions are well known in the art. For example, increasedcholesterol efflux from arterial lesions can be measured using the mousemodel of atherosclerosis described in Lawn et al., Nature, 360: 670-672(1992)), as well as any of the other known models. Using the LDLreceptor knockout mouse described in Lawn et al., cholesterol efflux canbe measured before and after administration of the ABC1 vector. Fattystreak lesion size in groups of animals fed an atherogenic diet can bemeasured by oil-red O staining of aortic sections as described in Lawnet al. A reduction in size of fatty streak lesions in the groupreceiving the ABC1 expression vector compared to a group receiving acontrol vector indicates an increase in cholesterol efflux from thelesions. In humans, the size of atherosclerotic lesions found inarterial walls can be measured using, for example, angiography andnon-invasive ultrasound methods.

Alternatively, cholesterol efflux can be measured by obtaining a bloodsample and measuring the level of ABC1 mRNA or ABC1 protein in themacrophage cells of a subject before and after administration of arecombinant ABC1 expression vector. Routine assays can be performed todetermine the correlation between increasing ABC1 mRNA concentrationsand cholesterol efflux. Likewise, assays can be performed to correlatethe amount of ABC1 protein with the amount of cholesterol efflux. Usingsuch correlation data, an observed increase in ABC1 mRNA or ABC1 proteinin the cells of a subject following administration of a recombinant ABC1expression vector can be used to indicate an increase in cholesterolefflux. ABC1 mRNA and ABC1 protein levels can be measured using theassays described herein and other well-known techniques for mRNA andprotein quantitation. Therapeutic dosages and formulations are discussedin further detail below.

There are available to one skilled in the art multiple viral andnon-viral methods suitable for introduction of a nucleic acid moleculeinto a target cell. For example, viral delivery vectors suitable forgene therapy include, but are not limited to, adenovirus, herpes simplexvirus, pox virus (i.e., vaccinia), hepatitis virus, parvovirus,papovavirus, alphavirus, coronavirus, rhabdovirus, papilloma virus,adeno-associated virus (AAV), polio virus, and RNA viruses, such as aretroviruses and Sindbis virus. The ABC1 polynucleotide can also bedelivered using a non-viral delivery system, such as naked DNA delivery(direct injection), receptor-mediated transfer (DNA-ligand complexes),electroporation, adenovirus-ligand-DNA complexes, calcium phosphate(CaPO₄) precipitation, microparticle bombardment (gene gun techniques),liposome-mediated transfer, and lipofection.

Genetic modification of a cell may be accomplished using one or moretechniques well known in the gene therapy field (Mulligan, R., 1993,Science, 260 (5110): 926-32). Gene therapy materials and methods caninclude inducible promoters, tissue-specific enhancer-promoters, DNAsequences designed for site-specific integration, DNA sequences capableof providing a selective advantage over the parent cell, labels toidentify transformed cells, negative selection systems and expressioncontrol systems (safety measures), cell-specific binding agents (forcell targeting), cell-specific internalization factors, andtranscription factors to enhance expression by a vector as well asmethods of vector manufacture. Examples of methods and materials for thepractice of gene therapy techniques are described in U.S. Pat. Nos.4,970,154 (involving electroporation techniques), 5,679,559 (describinga lipoprotein-containing system for gene delivery), 5,676,954 (involvingliposome carriers), 5,593,875 (describing methods for calcium phosphatetransfection), and 4,945,050 (describing a process wherein biologicallyactive particles are propelled at cells at a speed whereby the particlespenetrate the surface of the cells and become incorporated into theinterior of the cells), and PCT Pub. No. WO 96/40958 (involving nuclearligands).

Adenoviral vectors have proven especially useful for gene transfer intoeukaryotic cells (Rosenfeld, M., et al., Science, 252: 431-4 (1991);U.S. Pat. No. 5,631,236). The first trial of Ad-mediated gene therapy inhuman was the transfer of the cystic fibrosis transmembrane conductanceregulator (CFTR) gene to lung (Crystal, R., et al., 1994, Nat. Genet., 8(1): 42-51). Experimental routes for administrating recombinant Ad todifferent tissues in vivo have included intratracheal instillation(Rosenfeld, M., et al., 1992, Cell, 68 (1): 143-55) injection intomuscle (Quantin, B., et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89(7): 2581-4), peripheral intravenous injection (Herz, J., and Gerard,R., 1993, Proc. Natl. Acad. Sci. U.S.A., 90 (7): 2812-6) andstereotactic inoculation to brain (Le Gal La Salle, G., et al., 1993,Science, 259 (5097): 988-90). The adenoviral vector, then, is widelyavailable to one skilled in the art and is suitable for use in thepresent invention.

Adeno-associated virus (AAV) has recently been introduced as a genetransfer system with potential applications in gene therapy. Wild-typeAAV demonstrates high-level infectivity, broad host range andspecificity in integrating into the host cell genome (Hermonat, P., andMuzyczka, N., 1984, Proc. Natl. Acad. Sci. U.S.A., 81 (20): 6466-70).Herpes simplex virus type-1 (HSV-1) is a preferred vector system(Geller, A., et al., 1991, Trends Neurosci., 14 (10): 428-32; Glorioso,J., et al., 1995, Mol. Biotechnol., 4 (1): 87-99; Glorioso, J., et al.,1995, Annu. Rev. Microbiol., 49: 675-710). Vaccinia virus, of thepoxvirus family, has also been developed as an expression vector (Smith,G., and Moss, B., 1983, Gene, 25 (1): 21-8; Moss, B., 1992,Biotechnology, 20: 345-62; Moss, B., 1992, Curr. Top. Microbiol.Immunol., 158: 25-38). Each of the above-described vectors is widelyavailable to one skilled in the art and would be suitable for use in thepresent invention.

Preferably, the viral delivery system utilizes a retroviral vector.Retroviral vectors are capable of infecting a large percentage of thetarget cells and integrating into the cell genome (Miller, A., andRosman, G., Biotechniques, 7(9): 980-2, 984-6, 989-90 (1989); U.S. Pat.No. 5,672,510). Retroviruses were developed as gene transfer vectorsrelatively earlier than other viruses, and were first used successfullyfor gene marking and transducing the cDNA of adenosine deaminase (ADA)into human lymphocytes. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus, or is a lentiviral vector.An especially preferred retroviral vector is a lentiviral vector.Examples of retroviral vectors in which a single foreign gene can beinserted include, but are not limited to: Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A number ofadditional retroviral vectors can incorporate multiple genes. All ofthese vectors can transfer or incorporate a gene for a selectable markerso that transduced cells can be identified and generated.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. The helper plasmids are missinga nucleotide sequence that enables the packaging mechanism to recognizean RNA transcript for encapsitation. Thus, helper cell lines produceempty virions, since no genome is packaged. Suitable helper cell linesinclude, but are not limited to Ψ2, PA317 and PA12. If a retroviralvector is introduced into such cells in which the packaging signal isintact, but the structural genes are replaced by other genes ofinterest, the vector can be packaged and vector virion produced. Thevector virions produced by this method can then be used to infect atissue cell line, such as NIH 3T3 cells, to produce large quantities ofchimeric retroviral virions.

The vectors of the present invention may be constructed using standardrecombinant techniques widely available to one skilled in the art. Suchtechniques may be found in common molecular biology references such asMolecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991, Academic Press, SanDiego, Calif.), and PCR Protocols: A Guide to Methods and Applications(Innis, et al. 1990, Academic Press, San Diego, Calif.).

In order to obtain transcription of the ABC1 polynucleotide within atarget cell, a transcriptional regulatory region capable of driving geneexpression in the target cell must be utilized. The transcriptionalregulatory region preferably comprises a promoter and/or enhancer, whichis operatively linked to the ABC1 polynucleotide. The promoter can behomologous or heterologous to the ABC1 gene, provided that it is activein the cell or tissue-type into which the construct will be inserted.The transcriptional regulatory region chosen should drive high levelgene expression in the target cell. Preferably, a macrophage-specificpromoter, such as a scavenger receptor type A, matrix metalloproteinasepromoter (MMP-12), or macrophage-tropic lentivirus promoter (Fabunmi etal., Atherosclerosis, 148: 375-386 (1999)), is used. A particularlypreferred promoter is the 5′ region of the scavenger receptor type Agene, which contains a strong macrophage promoter that can be used todrive the transcription of the ABC1 gene. In addition, a means toincrease endogenous ABC1 polypeptide expression in a cell is to insertone or more enhancer elements into the promoter region, where theenhancer elements can serve to increase transcriptional activity of theABC1 gene. Similarly, the enhancer element(s) used is selected based onthe tissue in which one desires to activate the gene. Thus, for example,enhancer elements known to confer promoter activation in cells found inarterial tissue, especially macrophage cells, will be selected. Othertranscriptional regulatory regions suitable for use in the presentinvention include but are not limited to the human cytomegalovirus (CMV)immediate-early enhancer/promoter, the SV40 early enhancer/promoter, theJC polyomavirus promoter, the albumin promoter, PGK and the α-actinpromoter coupled to the CMV enhancer (Doll, R., et al., 1996, GeneTher., 3 (5): 437-47).

Other components of the vector construct may optionally include DNAmolecules designed for site-specific integration (e.g., endogenoussequences useful for homologous recombination), tissue-specificpromoters, DNA molecules capable of providing a selective advantage overthe parent cell, DNA molecules useful as labels to identify transformedcells, negative selection systems, cell specific binding agents (e.g.,for cell targeting), cell-specific internalization factors,transcription factors enhancing expression from a vector, and factorsenabling vector production.

In one embodiment, the vector can include targeting DNA forsite-specific integration. The targeting DNA is a nucleotide sequencethat is complementary (homologous) to a region of the gene of interest,for example, the ABC1 gene. Through homologous recombination, the DNAsequence to be inserted into the genome can be directed to the ABC1 geneby attaching it to the targeting DNA. DNA sequences for insertioninclude, for example, regions of DNA that may interact with or controlthe expression of an ABC1 polypeptide, e.g. flanking sequences. Thus,the expression of the desired ABC1 polypeptide is achieved not bytransfection of DNA that encodes the ABC1 gene itself, but rather by theuse of targeting DNA coupled with DNA regulatory segments that providethe endogenous gene sequence with recognizable signals for transcriptionof an ABC1 polypeptide (Sauer, Curr. Opin. Biotechnol., 5:521-27 (1994);Sauer, Methods Enzymol., 225:890-900 (1993)).

In yet other embodiments, regulatory elements can be included for thecontrolled expression of the ABC1 gene in the target cell. Such elementsare turned on in response to an appropriate effector. In this way, atherapeutic ABC1 polypeptide can be expressed when desired. Oneconventional control means involves the use of small molecule dimerizersor rapalogs to dimerize chimeric proteins which contain a smallmolecule-binding domain and a domain capable of initiating a biologicalprocess, such as a DNA-binding protein or transcriptional activationprotein (see PCT Pub. Nos. WO 96/41865, WO 97/31898, and WO 97/31899).The dimerization of the proteins can be used to initiate transcriptionof the transgene. Another suitable control means or gene switchesincludes the use of mifepristone (RU486), which is a progesteroneantagonist. The binding of a modified progesterone receptorligand-binding domain to the progesterone antagonist activatestranscription by forming a dimer of two transcription factors that thenpass into the nucleus to bind DNA. The ligand-binding domain is modifiedto eliminate the ability of the receptor to bind to the natural ligand.The modified steroid hormone receptor system is further described inU.S. Pat. No. 5,364,791 and PCT Pub. Nos. WO 96/40911 and WO 97/10337.Yet another control means uses a positive tetracycline-controllabletransactivator. This system involves a mutated tet repressor proteinDNA-binding domain (mutated tet R-4 amino acid changes which resulted ina reverse tetracycline-regulated transactivator protein, i.e., it bindsto a tet operator in the presence of tetracycline) linked to apolypeptide which activates transcription. Such systems are described inU.S. Pat. Nos. 5,464,758, 5,650,298, and 5,654,168. Additionalexpression control systems and nucleic acid constructs are described inU.S. Pat. Nos. 5,741,679 and 5,834,186.

Viral delivery vectors containing an ABC1 polynucleotide can be madetarget specific by altering the viral coat such that it contains aligand that is specific for another molecule found on the target cell.This will allow the vector to bind specifically to the desiredcell-type. The ligand can be any compound of interest that will bindspecifically to a molecule found on the target cell, such as acell-surface receptor. Preferably, the receptor is found exclusively ontarget cells and not on other cells. For example, ligands for scavengerreceptor A can be used to direct viral delivery vectors to macrophagecells. Alternatively, the viral coat can be altered such that itcontains an antibody or antibody fragment, such as Fab, or F(ab′)₂, thatrecognizes and binds to an antigenic epitope on the target cells. Theviral coat can be altered by inserting an additional polynucleotide thatencodes the ligand into the viral genome. Those of skill in the art willknow of other specific polynucleotide sequences which can be insertedinto the viral genome to allow target specific delivery of the vectorcontaining the ABC1 polynucleotide.

In addition, a naked ABC1 polynucleotide can be administered. ABC1polynucleotides and recombinant ABC1 expression vectors, such as thosedescribed above, can be administered as a pharmaceutical composition.Such a composition comprises an effective amount of an ABC1polynucleotide or recombinant ABC1 expression vector, as previouslydefined herein, and a pharmaceutically acceptable formulation agentselected for suitability with the mode of administration. Suitableformulation materials preferably are non-toxic to recipients at theconcentrations employed and are described herein below.

The pharmaceutical composition comprising an ABC1 polynucleotide or anABC1 recombinant expression vector may contain formulation materials formodifying, maintaining, or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption, or penetration of thecomposition. Suitable formulation materials include, but are not limitedto, amino acids (such as glycine, glutamine, asparagine, arginine, orlysine), antimicrobials, antioxidants (such as ascorbic acid, sodiumsulfite, or sodium hydrogen-sulfite), buffers (such as borate,bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids),bulking agents (such as mannitol or glycine), chelating agents (such asethylenediamine tetraacetic acid (EDTA)), complexing agents (such ascaffeine, polyvinylpyrrolidone, beta-cyclodextrin, orhydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,disaccharides, and other carbohydrates (such as glucose, mannose, ordextrins), proteins (such as serum albumin, gelatin, orimmunoglobulins), coloring, flavoring and diluting agents, emulsifyingagents, hydrophilic polymers (such as polyvinylpyrrolidone), lowmolecular weight polypeptides, salt-forming counterions (such assodium), preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide),solvents (such as glycerin, propylene glycol, or polyethylene glycol),sugar alcohols (such as mannitol or sorbitol), suspending agents,surfactants or wetting agents (such as pluronics; PEG; sorbitan esters;polysorbates such as polysorbate 20 or polysorbate 80; triton;tromethamine; lecithin; cholesterol or tyloxapal), stability enhancingagents (such as sucrose or sorbitol), tonicity enhancing agents (such asalkali metal halides—preferably sodium or potassium chloride—or mannitolsorbitol), delivery vehicles, diluents, excipients and/or pharmaceuticaladjuvants. See Remington's Pharmaceutical Sciences (18^(th) Ed., A. R.Gennaro, ed., Mack Publishing Company 1990).

The pharmaceutically active compounds (i.e. ABC1 polynucleotide or ABC1vector) can be processed in accordance with conventional methods ofpharmacy to produce medicinal agents for administration to patients,including humans and other mammals. Thus, the pharmaceutical compositioncomprising an ABC1 polynucleotide or an ABC1 recombinant expressionvector may be made up in a solid form (including granules, powders orsuppositories) or in a liquid form (e.g., solutions, suspensions, oremulsions). Solid dosage forms for oral administration may includecapsules, tablets, pills, powders, and granules. In such solid dosageforms, the active compound may be admixed with at least one inertdiluent such as sucrose, lactose, or starch. Such dosage forms may alsocomprise, as in normal practice, additional substances other than inertdiluents, e.g., lubricating agents such as magnesium stearate. In thecase of capsules, tablets, and pills, the dosage forms may also comprisebuffering agents. Tablets and pills can additionally be prepared withenteric coatings.

Liquid dosage forms for oral or parenteral administration may includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs containing inert diluents commonly used in the art, such aswater. Such compositions may also comprise adjuvants, such as wettingsweetening, flavoring, and perfuming agents. For example, a suitablecarrier for injection may be water, physiological saline solution, orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplarycarriers. Other exemplary pharmaceutical compositions comprise Trisbuffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, whichmay further include sorbitol or a suitable substitute.

The dosage regimen for treating a cardiovascular disease with acomposition comprising an ABC1 polynucleotide or ABC1 expression vectoris based on a variety of factors, including the type of cardiovasculardisease, the age, weight, sex, medical condition of the patient, theseverity of the condition, the route of administration, and theparticular compound employed. Thus, the dosage regimen may vary widely,but can be determined routinely using standard methods. For example, theamount of ABC1 polynucleotide or ABC1 expression vector to beadministered is an amount sufficient to increase cholesterol efflux fromthe cells of a mammalian subject. Such amount can be determined, forexample, by measuring the plasma HDL-cholesterol level of a subjectbefore and after administration of the ABC1 polynucleotide or ABC1expression vector. A sufficient amount of ABC1 polynucleotide or ABC1expression vector is an amount that increases the plasma HDL-cholesterollevel of a subject. Accordingly, the clinician can titer the dosage andmodify the route of administration to obtain the optimal therapeuticeffect. A typical dosage may range from about 0.1 g/kg to about 100mg/kg or more, depending on the factors mentioned above.

The frequency of dosing will depend upon the pharmacokinetic parametersof the ABC1 polynucleotide or vector in the formulation being used.Typically, a clinician will administer the composition until a dosage isreached that achieves the desired effect. The composition may thereforebe administered as a single dose, as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages may be ascertained through use ofappropriate dose-response data.

The cells of a mammalian subject may be transfected in vivo, ex vivo, orin vitro. Administration of an ABC1 polynucleotide or a recombinantvector containing an ABC1 polynucleotide to a target cell in vivo may beaccomplished using any of a variety of techniques well known to thoseskilled in the art. For example, U.S. Pat. No. 5,672,344 describes an invivo viral-mediated gene transfer system involving a recombinantneurotrophic HSV-1 vector. The above-described compositions of ABC1polynucleotides and recombinant ABC1 vectors can be transfected in vivoby oral, buccal, parenteral, rectal, or topical administration as wellas by inhalation spray. The term “parenteral” as used herein includes,subcutaneous, intravenous, intramuscular, intrasternal, infusiontechniques or intraperitoneally.

For oral administration, the pharmaceutical composition containing theABC1 polynucleotide or recombinant ABC1 vector may be in the form of,for example, a capsule, a tablet, a suspension, or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a given amount of DNA or viral vector particles. Forexample, these may contain an amount from about 10³-10¹⁵ viralparticles, preferably from about 10⁶-10¹² viral particles. A suitabledaily dose for a human or other mammal may vary widely depending on thecondition of the patient and other factors, but, once again, can bedetermined using routine methods.

The pharmaceutical composition containing the ABC1 DNA or recombinantABC1 vector can also be administered rectally. Suitable suppositoriesfor rectal administration of the vector can be prepared by mixing thevector with a suitable non-irritating excipient such as cocoa butter andpolyethylene glycols that are solid at ordinary temperatures but liquidat the rectal temperature and will therefore melt in the rectum andrelease the vector.

A pharmaceutical composition also can be formulated for inhalation. Forexample, an ABC1 polynucleotide or vector may be formulated as a drypowder for inhalation. Also, ABC1 polynucleotide or vector inhalationsolutions can be formulated with a propellant for aerosol delivery. Inyet another embodiment, solutions may be nebulized.

The pharmaceutical composition containing the ABC1 DNA or recombinantABC1 vector can also be injected. Injectable preparations, such assterile injectable aqueous or oleaginous suspensions, may be formulatedaccording to the known methods using suitable dispersing or wettingagents and suspending agents. The sterile injectable preparation mayalso be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. A particularly suitable carrier for parenteral injectionis sterile distilled water in which an ABC1 polynucleotide or vector isformulated as a sterile, isotonic solution, properly preserved. Amongthe other acceptable carriers and solvents that may be employed areRinger's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employed,including synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables. Yet anotherpreparation can involve the formulation of the desired ABC1 moleculewith an agent, such as injectable microsperes, bio-erodible particles,polymeric compounds, beads or liposomes, that provides for thecontrolled or sustained release of the ABC1 product (see, e.g.PCT/US93/00829; Eppstein et al., Proc. Natl. Acad. Sci., 82: 3688-3692(1985)).

Also, the pharmaceutical composition containing the ABC1 DNA orrecombinant ABC1 vector can administered topically. For topicaladministration, the vector may comprise from 0.001% to 10% w/w, e.g.,from 1% to 2% by weight of the formulation, although it may comprise asmuch as 10% w/w, but preferably not more than 5% w/w, and morepreferably from 0.1% to 1% of the formulation. Formulations suitable fortopical administration include liquid or semi-liquid preparationssuitable for penetration through the skin (e.g., liniments, lotions,ointments, creams, or pastes) and drops suitable for administration tothe eye, ear, or nose. A suitable topical dose of active ingredient of avector of the present invention is administered one to four, preferablytwo or three times daily.

While the nucleic acids and/or vectors of the invention can beadministered as the sole active pharmaceutical agent, they can also beused in combination with one or more vectors of the invention or otheragents. When administered as a combination, the therapeutic agents canbe formulated as separate compositions that are given at the same timeor different times, or the therapeutic agents can be given as a singlecomposition.

In another embodiment of the present invention, a target cell istransfected in vivo by implantation of a “producer cell line” inproximity to the target cell population (Culver, K., et al., 1994, Hum.Gene Ther., 5 (3): 343-79; Culver, K., et al., Cold Spring Harb. Symp.Quant. Biol., 59: 685-90; Oldfield, E., 1993, Hum. Gene Ther., 4 (1):39-69). The producer cell line is engineered to produce a viral vectorcontaining the ABC1 polynucleotide and to release its viral particles inthe vicinity of the target cells, i.e. preferably macrophage cells foundin atherosclerotic lesions. A portion of the released viral particlescontact the target macrophage cells and infect those cells, thusdelivering an ABC1 polynucleotide to the target macrophage cell.Following infection of the target cell, expression of the ABC1polynucleotide occurs, providing the macrophage cell with functionalABC1 protein.

In another embodiment, the invention provides a method of treating acardiovascular disease by the ex vivo introduction of an ABC1polynucleotide or recombinant ABC1 expression vector. In such instances,cells, tissues, or organs that have been removed from the patient areexposed to ABC1 compositions after which the cells, tissues, or organsare subsequently implanted back into the patient. For example, onemethod includes the removal of a blood sample from a subject withcardiovascular disease, enriching the sample for monocytes, andcontacting the isolated monocytes with a recombinant expression vectorcontaining the ABC1 polynucleotide and, optionally, a target specificgene. Optionally, the monocyte cells can be treated with a growthfactor, such as GM-CSF, to stimulate cell growth, before reintroducingthe cells into the subject. When reintroduced, the cells willspecifically target the cell population from which they were originallyisolated. In this way, the transport activity of the ABC1 polypeptidemay be used to promote cholesterol efflux in a subject.

Another method of ex vivo administration involves introducing the ABC1polynucleotide or recombinant ABC1 vector into the mammalian subject bymeans of skin transplants of cells containing the virus. Preferably, aretrovius used for this method of administration. Long term expressionof foreign genes in implants, using cells of fibroblast origin, may beachieved if a strong housekeeping gene promoter is used to drivetranscription. For example, the dihydrofolate reductase (DHFR) genepromoter may be used. Cells such as fibroblasts, can be infected withvirions containing a retroviral construct containing the ABC1polynucleotide together with a gene which allows for specific targeting,such as scavenger receptor A, and a strong promoter. The infected cellscan be embedded in a collagen matrix that can be grafted into theconnective tissue of the dermis in the recipient subject. As theretrovirus proliferates and escapes the matrix it will specificallyinfect the target cell population. In this way the transplantationresults in increased amounts of cholesterol efflux activity in cellsmanifesting the transport disorder.

In another embodiment, the recombinant expression vector comprising theABC1 polynucleotide can be administered using in vitro techniques, suchas described in U.S. Pat. No. 5,399,346. For example, an ABC1polypeptide can be delivered by implanting certain cells that have beengenetically engineered, using methods such as those described herein, toexpress the ABC1 polypeptide. In order to minimize a potentialimmunological reaction in patients being administered an ABC1polypeptide, as may occur with the administration of a polypeptide of aforeign species, it is preferred that the natural cells producing ABC1polypeptide be of human origin and produce human ABC1 polypeptide. Thus,it is preferred that the recombinant cells producing ABC1 polypeptide betransformed with an expression vector containing a gene encoding a humanABC1 polypeptide. The cells can be autologous or heterologous.Optionally, the cells can be immortalized. In order to decrease thechance of an immunological response, the cells may be encapsulated toavoid infiltration of surrounding tissues. The encapsulation materialsare typically biocompatible, semi-permeable polymeric enclosures ormembranes that allow the release of the protein product(s) but preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissues. The transfected cellscan be administered to a patient using the above-described methods.

Techniques for the encapsulation of living cells are known in the art,and the preparation of the encapsulated cells and their implantation inpatients may be routinely accomplished. For example, Baetge et al. (PCTPub. No. WO 95/05452 and PCT/US94/09299) describe membrane capsulescontaining genetically engineered cells for the effective delivery ofbiologically active molecules. The capsules are biocompatible and areeasily retrievable. The capsules encapsulate cells transfected withrecombinant DNA molecules comprising DNA sequences coding forbiologically active molecules operatively linked to promoters that arenot subject to down regulation in vivo upon implantation into amammalian host. The devices provide for the delivery of the moleculesfrom living cells to specific sites within a recipient. In addition, seeU.S. Pat. Nos. 4,892,538; 5,011,472; and 5,106,627. A system forencapsulating living cells is described in PCT Pub. No. WO 91/10425(Aebischer et al). See also, PCT Pub. No. WO 91/10470 (Aebischer etal.); Winn et al., 1991, Exper. Neurol. 113:322-29; Aebischer et al.,1991, Exper. Neurol. 111:269-75; and Tresco et al., 1992, ASAIO38:17-23.

Another delivery system for polynucleotides encoding ABC1 is a“non-viral” delivery system. Techniques that have been used or proposedfor gene therapy include DNA-ligand complexes, adenovirus-ligand-DNAcomplexes, direct injection of DNA, CaPO₄ precipitation, gene guntechniques, electroporation, lipofection, and colloidal dispersion(Mulligan, R., 1993, Science, 260 (5110): 926-32). Any of these methodsare widely available to one skilled in the art and would be suitable foruse in the present invention. Other suitable methods are available toone skilled in the art, and it is to be understood that the presentinvention may be accomplished using any of the available methods oftransfection. Several such methodologies have been utilized by thoseskilled in the art with varying success (Mulligan, R., 1993, Science,260 (5110): 926-32).

Preferably, the non-viral delivery system is a colloidal dispersionsystem. Colloidal dispersion systems include macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Thepreferred colloidal system of this invention is a liposome, which is anartificial membrane vesicle useful as delivery vehicles in vitro and invivo. Liposomes are self-assembling, colloidal particles in which alipid bilayer, composed of amphiphilic molecules such as phosphatidylserine or phosphatidyl choline, encapsulates a portion of thesurrounding media such that the lipid bilayer surrounds a hydrophilicinterior. Unilammellar or multilammellar liposomes can be constructedsuch that the interior contains a desired chemical, drug, or, as in theinstant invention, an isolated DNA molecule. For example, it has beenshown that large unilamellar vesicles (LUV), which range in size from0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules, such as RNA, DNA and intact virions.Once encapsulated within the aqueous interior, these macromolecules canbe delivered to mammalian cells in a biologically active form (Fraley,R. and Papahadjopoulos, D. 1981, Trends Biochem. Sci., 6: 77-80). Inorder for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of thegenes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, R., et al., 1988, Biotechniques, 6 (7): 682-90).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations. Examples of lipids useful in liposomeproduction include phosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidyletha-nolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes has been classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand, such as apolyclonal or monoclonal antibody, sugar, glycolipid, or protein, or bychanging the composition or size of the liposome in order to achievetargeting to organs and cell types other than the naturally occurringsites of localization. Preferably, the ligand is a polyclonal ormonoclonal antibody which can be used to target liposomes to specificcell-surface ligands. The ligand can also be an antibody fragment, suchas Fab, or F(ab′)₂, as long as it binds efficiently to an the antigenicepitope on the target cells. Preferably, the antibody or antibodyfragment recognizes an antigen that is found exclusively on the targetcell. For example, certain antigens expressed specifically on macrophagecells, such as scavenger receptor A, may be exploited for the purpose oftargeting antibody-ABC1 liposomes directly to a macrophage cell.

A number of procedures can be used to covalently attach eitherpolyclonal or monoclonal antibodies to a liposome bilayer. Also, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. In addition, various linking groups can be used forjoining the lipid chains to the targeting ligand.

Studies presented herein showed that ligands for nuclear receptors wereable to effect an increase in cholesterol efflux. Accordingly, inanother embodiment, the present invention provides a method suitable forincreasing cholesterol efflux from cells in a mammalian subjectcomprising the step of administering to the mammalian subject at leastone ligand for a nuclear receptor in an amount sufficient to increasecholesterol efflux from said cells. A pharmaceutical compositioncomprising a ligand for a nuclear receptor can prepared and administeredusing the above-described methods for formulating and administeringpharmaceutical compositions. A sufficient amount of a nuclear receptorligand is the amount of ligand that increases cholesterol efflux. Suchamount can be determined by measuring the cholesterol efflux before andafter administration of the ligand at various dosages and determiningthe dose that effects an increase in cholesterol efflux. The cholesterolefflux can be measured using assays previously described.

Nuclear receptors are ligand-activated transcription factors that play acritical role in vertebrate development and adult physiology bytransducing the effects of small, lipophilic hormones intotranscriptional responses. Several families of nuclear receptors exist,including peroxisome proliferator-activated receptors (PPARs), liver Xreceptors (LXR), retinoid X receptor (RXR), the farnesoid X receptor(FXR), and the steroid and xenobiotic receptor (SXR). The PPAR familycomprises the three closely related gene products PPARα, PPARγ, andPPARβ/δ. PPARα has been implicated as a key regulator of intra- andextracellular lipid metabolism. When bound to fatty acids, PPARαstimulates the proliferation of peroxisomes and induces the synthesis ofseveral enzymes involved in the β-oxidation of fatty acids. The PPARαreceptor is also the molecular target for the fibrates, drugs that areprescribed for the reduction of high triglyceride levels (Isseman etal., Nature, 347, 645 (1990)). Fibrates act as PPAR ligands to regulatethe transcription of a large number of genes that affect lipoprotein andfatty acid metabolism. In addition, PPARγ ligands, such as compoundsbelonging to the class of thiazolidinediones compounds, have been shownto increase HDL and reduce triglyceride levels in humans.

LXR is an oxysterol receptor that regulates the catabolism of excesscholesterol. LXRα has been shown to bind as a heterodimer with RXR to aDNA response element in the CYP7a gene, which encodes the enzymeresponsible for the rate-limiting step in the conversion of cholesterolto bile acids. Studies have shown that mice lacking LXRα accumulateenormous amounts of cholesterol esters in their livers when fed acholesterol-rich diet, due to their inability to increase CYP7a genetranscription in response to dietary cholesterol (Peet et al., Cell, 93:693 (1998)). Further studies with LXRα null mice show that LXRα is alsoinvolved in the regulation of several other genes that participate incholesterol and fatty acid homeostasis. The biological role of a closelyrelated nuclear receptor, LXRβ, which is expressed in several tissuesand is activated by the same oxysterols as LXRα, remains unclear (Songet al., Proc. Natl. Acad. Sci., 91: 10809 (1994); Seol, Mol.Endocrinol., 9: 72 (1995)).

FXR, which is evolutionarily related to LXRα, is also involved incholesterol homeostasis. Like LXRα, FXR functions as a heterodimer withthe RXR receptor (Schwartz et al., Curr. Opin. Lipidol., 9: 113 (1998);Vlahcevic et al., Gastroenterology, 113: 1949 (1997)). FXR is activatedby the synthetic retinoid TTNPB and superphysiological concentrations ofall-trans retinoic acid (Zavacki et al., Proc. Natl. Acad. Sci., 94:7909 (1997)). Recent studies indicate that FXR is a nuclear bile acidreceptor. First, FXR is abundantly expressed in tissues through whichbile acids circulate, including the liver, intestine, and kidney (Seolet al., Mol. Endocrinol., 9: 72 (1995); Forman et al., Cell, 81: 687(1995)). Also, FXR has recently been shown to serve as a receptor forphysiological concentrations of several bile salts, among whichchenodeoxycholic acid (CDCA) is the most potent (Kliewer et al.,Science, 284: 757-284 (1999); Makishima et al., Science, 284: 1362-1363(1999)). CDCA is known to regulate the expression of several genes thatparticipate in bile salt homeostasis, including those encoding CYP7a andthe intestinal bile acid-binding protein.

As described in detail in Example 13, ligands for LXR, RXR, and PPARnuclear receptors were shown to increase apoAI-induced cholesterolefflux in cholesterol-loaded mouse macrophage cells. For example,administration of 9 cis-retinoic acid (30 ng/ml) produced approximatelya 3-fold increase in apoAI-induced cholesterol efflux in these cells.Similarly, administration of 22(R)-hydroxycholesterol (5 μg/ml) producedapproximately a 3-fold increase in apoAI-induced cholesterol efflux.Cells that received fenfibrate (3 μg/ml) produced an approximate 2-foldincrease in cholesterol efflux. These results indicate that nuclearreceptors may be modulated to increase the rate ofapolipoprotein-mediated cholesterol efflux from macrophages.Furthermore, as described in Example 13, 9-cis-RA mediated cholesterolefflux from macrophage cells in a dose-dependent manner. Other nuclearreceptor activators, such as bezafibrate, were shown to increasecholesterol efflux (data not shown).

Accordingly, in the method for increasing cholesterol efflux byadministering a nuclear receptor ligand, the ligand is preferablyselected from the group consisting of LXR, RXR, PPAR, FXR, and SXRnuclear receptor ligands. In the preferred embodiment wherein a LXRligand is used to increase cholesterol efflux, the ligand is morepreferably selected from the group consisting of 20(S)hydroxycholesterol, 22(R) hydroxycholesterol, 24-hydroxycholesterol,25-hydroxycholesterol, and 24(S), 25 epoxycholesterol LXR ligands. Mostpreferably, the LXR ligand is 20(S) hydroxycholesterol. In the preferredembodiment wherein a RXR ligand is used to increase cholesterol efflux,the ligand is more preferably selected from the group consisting of9-cis retinoic acid, retinol, retinal, all-trans retinoic acid, 13-cisretinoic acid, acitretin, fenretinide, etretinate, CD 495, CD564, TTNN,TTNNPB, TTAB, LGD 1069. Most preferably, the RXR ligand is 9-cisretinoic acid. In another preferred embodiment wherein a PPAR ligand isused to increase cholesterol efflux, the ligand is preferably selectedfrom the class of thiazolidinedione compounds.

In another preferred embodiment, more than one nuclear receptor ligandis administered to the mammalian subject to increase cholesterol efflux.Preferably, when two or more nuclear receptor ligands are administeredto a subject, the ligands are an LXR and an RXR ligand. More preferably,the nuclear receptor ligands are 20(S) hydroxycholesterol and 9-cisretinoic acid.

In still another embodiment, the present invention provides a methodsuitable for increasing cholesterol efflux from cells in a mammaliansubject comprising the step of administering to the mammalian subject aneicosanoid in an amount sufficient to increase cholesterol efflux. Apharmaceutical composition comprising an eicosanoid can be prepared andadministered using the above-described methods for formulating andadministering pharmaceutical compositions. A sufficient amount ofeicosanoid is the amount that increases cholesterol efflux. Such amountcan be determined by measuring the cholesterol efflux before and afteradministration of the eicosanoid at various dosages and determining thedose that effects an increase in cholesterol efflux. The cholesterolefflux can be measured using assays and methods previously described.

As described in Example 14, eicosanoids were shown to increaseapoAI-induced cholesterol efflux in cholesterol-loaded mouse macrophagecells. For example, administration of PGI2 (25 nm) producedapproximately a 2-fold increase in apoAI-induced cholesterol efflux inthese cells. Likewise, administration of PGE1 (25 nM) producedapproximately a 3-fold increase in apoAI-induced cholesterol efflux.These results demonstrate that eicoasnoids can increase the rate ofapolipoprotein-mediated cholesterol efflux from macrophages.Accordingly, in a preferred embodiment, the eicosanoid is selected fromthe group consisting of prostaglandin E2, prostacyclin (prostaglandinI2), and prostaglandin J2 eicosanoids.

Methods and Compounds for Increasing ABC1 Expression/Activity

Given that ABC1 functions to promote cholesterol efflux, one way toincrease cholesterol efflux is to increase the cellular expression ofABC1. Accordingly, the present invention also provides methods suitablefor increasing cholesterol efflux from cells in a mammalian subject byadministering to the mammalian subject a therapeutic amount of acompound that increases the expression of ABC1 in said cells. Atherapeutic amount of compound is the amount of compound that increasesABC1 expression. Such amount can be determined by measuring the geneexpression of ABC1 before and after administration of the compound atvarious dosages and determining the dose that effects an increase inABC1 gene expression. The ABC1 gene expression can be measured byobtaining a blood sample from the subject, separating the monocytepopulation, and determining the concentration of ABC1 mRNA using methodsknown in the art and described herein, such as RT-PCR.

In one preferred embodiment, the method comprises administering a cAMPanalogue to increase the gene expression of ABC1. As shown in FIG. 8,cAMP increases the expression of ABC1 mRNA in normal fibroblast cellsapproximately 10-fold. Preferably, the cAMP analogue is selected fromthe group consisting of 8-bromo cAMP, N6-benzoyl cAMP, and 8-thiomethylcAMP. In another preferred embodiment, the method comprisesadministering a compound that increases the synthesis of cAMP toincrease the gene expression of ABC1. Preferably, the compound isforskolin. In yet another preferred embodiment, the method comprisesadministering a compound that inhibits the breakdown of cAMP to increasethe gene expression of ABC1. An example of such a compound is aphosphodiesterase inhibitor. Preferably, the phosphodiesterase inhibitoris selected from the group consisting of rolipram, theophylline,3-isobutyl-1-methylxanthine, R020-1724, vinpocetine, zaprinast,dipyridamole, milrinone, aminone, pimobendan, cilostamide, enoximone,peroximone, and vesnarinone phosphodiesterase inhibitors.

In another preferred embodiment, the method comprises administering tothe mammalian subject a ligand for a nuclear receptor in an amountsufficient to increase the gene expression of ABC1. As described inExample 17 and shown in FIG. 12, ligands for nuclear receptors canup-regulate the gene expression of ABC1. Transfection studies usingpAPR1, which contains a luciferase reporter gene under the control ofthe ABC1 promoter, showed that the ABC1 promoter was activated in thepresence of ligands for LXR and RXR nuclear receptors. Specifically,macrophage cells transfected with pAPR1 produced a 19-fold increase inluciferase reporter activity in the presence of 200H-chol, a 16-foldincrease in luciferase activity in the presence of 9-cis RA, and a280-fold increase in luciferase activity in the presence of both ligandscompared with EtOH control. The results indicate that both sterols andretinoids elicit a strong transcription response from the ABC1 promoter.Further, there is an apparent synergistic effect between the two classesof compounds, as can be seen by the dramatic increase in luciferaseactivity found in cells treated with both ligands. In accordance withthe inventive method, preferably, the ligand is selected from the groupconsisting of LXR, RXR, PPAR, FXR, and SXR ligands.

In addition to increasing cellular levels of ABC1 protein, reversecholesterol transport can be promoted by enhancing the activity of ABC1protein. Thus, in another embodiment, the present invention provides amethod suitable for increasing cholesterol efflux from cells in amammalian subject comprising the step of administering to the mammaliansubject a therapeutic amount of a compound that increases ABC1 activityin an amount sufficient to increase cholesterol efflux. A pharmaceuticalcomposition comprising such a compound can prepared and administeredusing the above-described methods for formulating and administeringpharmaceutical compositions. A therapeutic amount of compound is theamount of compound that increases cholesterol efflux. Such amount can bedetermined by measuring the cholesterol efflux before and afteradministration of the compound at various dosages and determining thedose that effects an increase in cholesterol efflux using methodspreviously described. To determine whether an increase in cholesterolefflux is due to an increase in ABC1 activity, the amount of ABC1protein present in a cell sample before and after administration of thecompound is determined, using methods described herein (see Example 11).For both measurements (i.e. pre- and post-administration of thecompound), the amount of cholesterol efflux activity is divided by theconcentration of ABC1 protein to determine the amount of cholesterolactivity found in a standard concentration of ABC1 protein. An observedincrease in cholesterol activity standardized to protein concentrationindicates that the increase is due to an increase in ABC1 activity.

Methods for Identifying Therapeutic Compounds

Another aspect of the present invention relates to methods for screeninga compound to determine whether the compound modulates (i.e.,up-regulates or down-regulates) the gene expression of ABC1. Suchcompounds may be useful in the development of therapeutic compounds thatincrease ABC1 expression and thereby promote cholesterol efflux andraise blood levels of HDL-cholesterol. Accordingly, methods foridentifying compounds that may be useful in the treatment ofcardiovascular disease are provided. In one embodiment, the presentinvention provides a method for screening a test compound for ABC1expression modulating activity comprising the steps of: (a) operativelylinking a reporter cDNA with an expression modulating portion of themammalian ABC1 gene to produce a recombinant reporter construct; (b)transfecting the recombinant reporter construct into a population ofhost cells; (c) assaying the level of reporter gene expression in asample of the transfected host cells; (d) contacting the transfectedhost cells with the test compound being screened; (e) assaying the levelof reporter gene expression in a sample of the transfected host cellsafter contact with the test compound; and (f) comparing the relativechange in the level of reporter gene expression caused by exposure tothe test compound, thereby determining the ABC1 expression modulatingactivity.

First, a recombinant reporter construct comprising a heterologousreporter gene operatively linked to an expression modulating portion ofthe ABC1 gene is constructed. The ABC1 expression modulatingpolynucleotide and reporter gene can be inserted into a vector usingwell-known ligation and cloning techniques, such as those describedherein and in standard laboratory manuals, including Davis et al., BasicMethods in Molecular Biology (1986); Sambrook et al., Molecular Cloning:A Laboratory Manual, 2^(nd) Ed., (Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989)); and Ausubel et al. eds., Current Protocols inMolecular Biology, (Wiley and Sons (1994)). Alternatively, the ABC1expression modulating polynucleotide can be inserted into a commerciallyavailable reporter construct, such as those previously described. Anyvector suitable for ABC1 polynucleotide and reporter gene insertion canbe used. The chosen vector should be functional in the particular hostcell employed. Preferably, the vector is compatible with mammalian hostcells.

Preferably, the expression modulating portion of the ABC1 gene is the 5′flanking region of ABC1, containing ABC1 promoter activity. In onepreferred embodiment, the expression modulating portion of the ABC1 genecomprises SEQ ID NO: 3. In another preferred embodiment, the expressionmodulating portion of the ABC1 gene comprises nucleotides 1-1532,1080-1643, 1181-1643, 1292-1643, 1394-1643, or 1394-1532 of SEQ ID NO:3. Also, preferably the heterologous reporter is selected from the groupconsisting of polynucleotides that encode the luciferase,β-galactosidase, chloramphenicol acetyl transferase, and greenfluorescent proteins. More preferably, the heterologous reporter is apolynucleotide that encodes the luciferase protein. In a particularlypreferred embodiment, the recombinant reporter construct is pAPR1, shownin FIG. 11.

Next, the recombinant reporter construct is transfected into apopulation of host cells. The recombinant reporter construct can beintroduced into the host cells using any of the previously describedtransfection methods, as well as the methods described in Examples 8 and15. For example, the reporter construct can be transfected using calciumphosphate transfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,or any of the other known and described methods (see, e.g., Davis etal., Basic Methods in Molecular Biology (1986)). The host cell can beany cell that, when cultured under appropriate conditions, synthesizes areporter polypeptide, which can be subsequently measured. Preferably,the host cell is a mammalian host cell. More preferably, the mammalianhost cell is a macrophage, fibroblast, hepatic, or intestinal cell. Mostpreferably, the host cell is selected from the group consisting of RAW264.7 cells, Thp-1 cells, and HepG2 cells. The concentration of reporterconstruct and duration of the transfection can vary, depending on thetransfection method and the type and concentration of host cell used.Determination of the appropriate concentration of reporter construct andtransfection time is well within the skill of the ordinary artisan.

Following transfection, a sample of transfected host cells that was notexposed to the test compound is assayed to determine the level ofreporter gene expression. The level of reporter gene expression found inthe sample of unexposed transfected host cells provides a controlmeasurement. The transfected host cells are lysed and the level ofreporter gene expression is assayed using any of the methods well-knownin the art. The assays used to measure the level of reporter geneexpression differ, depending on the reporter construct used in thetransfections. For example, if a luciferase reporter construct is used,the luciferase activity of the cell lysate is measured as light unitsusing a luminometer, as described in Example 15.

A different sample of the transfected host cells is contacted with thetest compound being screened and the level of reporter gene expressionfound in these cells is subsequently measured. Preferably, thetransfected host cells are contacted with the test compound for about4-48 hours. More preferably, the transfected host cells are contactedwith the test compound for about 8-36 hours. Even more preferably, thetransfected host cells are contacted with the test compound for about 24hours. The same assay used to measure the level of reporter geneexpression in unexposed (control) cells should be used to measure thelevel of reporter gene expression in the cells exposed to the testcompound.

Finally, the level of reporter gene expression found in unexposedcontrol cells is compared with the level of reporter gene expressionfound in cells exposed to the test compound to determine whether thetest compound has ABC1 expression modulating activity. If the level ofgene expression in both cell samples are the same or about the same, thetest compound does not modulate ABC1 gene expression. A higher level ofreporter gene expression in cells exposed to the test compound relativeto the level of reporter gene expression found in control cells,indicates that the test compound up-regulates the gene expression ofABC1. A lower level of reporter gene expression in cells exposed to thetest compound relative to the level of reporter gene expression found incontrol cells, indicates that the test compound down-regulates the geneexpression of ABC1.

Another aspect of the present invention relates to methods for screeninga test compound to determine whether the compound promotes ABC1-mediatedcholesterol efflux. Such method comprises: (a) assaying the level ofcholesterol efflux in a sample of mammalian cells maintained in cultureto determine a control level of cholesterol efflux, (b) contacting themammalian cells with the test compound being screened; (c) assaying thelevel of cholesterol efflux in a sample of cells after contact with thetest compound; and (d) assaying the level of ABC1-dependent cholesterolefflux in a sample of cells after contact with the test compound,thereby determining whether the test compound promotes ABC1-mediatedcholesterol efflux from cells in culture.

The level of cholesterol efflux in a sample of cultured cells can bedetermined using methods known in the art and described herein (seeExample 1). Any mammalian cells that can be maintained in culture can beused to measure cholesterol efflux. The cells can be derived fromprimary cultures or from immortalized cell lines. For convenience, cellscan be immortalized by transfecting them with amphotropic retrovirusescontaining vectors with inserts of human papillomavirus 16, oncogenes E6and E7, and a selectable marker gene, as described in Example 1.Preferably, the cultured cells are fibroblast, macrophage, hepatic, orintestinal cells. More preferably, the cultured cells are RAW 264.7cells.

The level of cholesterol efflux in a sample of cells that has not beencontacted with the test compound is measured to obtain a control levelof cholesterol efflux. In addition, the level of cholesterol efflux in asample of mammalian cells that has been contacted with the test compoundis measured to determine the amount of cholesterol efflux affected bythe test compound. Also, the level of ABC1-mediated cholesterol effluxin a sample of mammalian cells that has been contacted with the testcompound is measured to determine the amount of ABC1-mediatedcholesterol efflux affected by the test compound. Preferably, the cellsare contacted with the test compound for about 8-24 hours beforecholesterol efflux or ABC1-mediated cholesterol efflux is assayed. Thelevel of ABC1-mediated cholesterol efflux can be assayed using ananti-ABC1 antibody that, upon binding, inhibits the activity of ABC1.Alternatively, the level of ABC1-mediated cholesterol efflux can beassayed using an anti-sense ABC1 polynucleotide that inhibits theexpression of ABC1. For example, the level of ABC1-mediated cholesterolefflux can be assayed using the anti-sense ABC1 polynucleotidecomprising SEQ ID NO: 57 (see Example 7). The cells should be contactedwith the anti-ABC1 antibody or anti-sense ABC1 polynucleotide at thesame time and for the same duration that it is contacted with the testcompound.

If the level of control cholesterol efflux is the same or about the sameas the level of cholesterol efflux found in cells contacted with thetest compound, the compound does not promote cholesterol efflux. Anincrease in the level of cholesterol efflux found in cells contactedwith test compound over the control level of cholesterol effluxindicates the amount of cholesterol efflux promoted by the testcompound. The difference between the cholesterol efflux found in cellscontacted with test compound alone and the cholesterol efflux found incells contacted with test compound and anti-ABC1 antibody or anti-senseABC1 polynucleotide indicates the amount of cholesterol efflux mediatedthrough ABC1. For example, if control level of cholesterol efflux is 1.0and the level of cholesterol efflux found in cells contacted with testcompound is 1.1, the test compound promotes cholesterol efflux by 10%.If the cholesterol efflux found in cells contacted with test compoundand anti-ABC1 antibody or anti-sense ABC1 polynucleotide is 1.0, theincrease in cholesterol efflux caused by the test compound is entirelyABC1-mediated.

Methods for Detecting Susceptibility to Coronary Heart Disease

The present invention also relates to methods for detecting thecomparative level of ABC1 gene or protein expression in a mammaliansubject, including a human subject. Given the role of ABC1 incholesterol efflux, the determination of a decreased level of ABC1 geneor protein expression in a mammalian subject relative to apre-determined standard level of ABC1 gene or protein expression can beused to indicate a susceptibility to coronary heart disease in thesubject. Accordingly, the present invention provides a method fordetecting the comparative level of ABC1 gene expression in a mammaliansubject comprising the steps of: (a) obtaining a test cell sample fromthe mammalian subject; (b) assaying the level of ABC1 mRNA expression inthe test cell sample; and (c) comparing the level of ABC1 mRNAexpression in the test cell sample with a pre-determined standard levelof ABC1 mRNA expression, thereby detecting the comparative level of ABC1gene expression in the mammalian subject.

A test cell sample is first obtained from a mammalian subject, includinga human subject. The test cell sample can be a blood sample, wherein themonocyte population has been enriched. Monocytes can be enriched usingwell-known cell separation procedures based on, for example, cell size,cell density or cell affinity. Next, the level of ABC1 mRNA expressionin the test cell sample is assayed. The level of ABC1 mRNA expressioncan be assayed using any of the well-known methods for mRNA preparationand detection, including the methods described herein at Examples 2 and9. For example, the concentration of ABC1 mRNA can be determined byreverse transcription polymerase chain reaction, northern blot analysis,or RNAse protection assay. The ABC1 mRNA concentration should bestandardized to the concentration of total mRNA found in the test cellsample. Finally, the ABC1 expression in the test cell sample is comparedwith a pre-determined standard level of ABC1 mRNA expression. Apre-determined standard level of ABC1 mRNA expression can be obtained bydetermining the average concentrations and distribution of ABC1 mRNAfound in cell samples taken from a representative population ofmammalian subjects, wherein the mammalian subjects are the same speciesas the subject from which the test cell sample was obtained, and whereinthe mammalian subjects do not have coronary heart disease, Tangierdisease, or other disease associated with low HDL-cholesterol and areconsidered to have cholesterol efflux activity within a normal range(i.e., as indicated by an HDL-cholesterol level within a normal range).The determination of a decreased level of ABC1 mRNA expression in thetest cell sample of a mammalian subject relative to the pre-determinedstandard level of ABC1 mRNA expression can be used to indicate asusceptibility to coronary heart disease in the mammalian subject.

Likewise, the detection of a decreased level of ABC1 protein can be usedto indicate decreased capacity for cholesterol efflux and asusceptibility to coronary heart disease. Accordingly, anotherembodiment of the present invention provides a method for detecting thecomparative level of ABC1 protein in a mammalian subject. Such methodcomprises the steps of: (a) obtaining a test cell sample from themammalian subject; (b) assaying the amount of ABC1 protein in the testcell sample; and (c) comparing the amount of ABC1 protein in the testcell sample with a pre-determined standard amount of ABC1 protein,thereby detecting the comparative level of ABC1 protein in the mammaliansubject.

The amount of ABC1 protein can be assayed using any of the well-knownmethods of measuring protein. Preferably, the amount of ABC1 protein ismeasured using an immunoassay. In one embodiment, the amount of ABC1protein is determined by (a) contacting the cell sample with apopulation of anti-ABC1 antibodies and (b) detecting the anti-ABC1antibodies associated with the cell sample. For example, the ABC1protein can be contacted with an antiserum raised against a syntheticpeptide corresponding to KNQTVVDAVLTSFLQDEKVKES (SEQ ID NO: 60) locatedat the C-terminus, as described in Example 11. The anti-ABC1 antibodiescan be detected using several methods known in the art, including, forexample, western blotting, immunoprecipitation, and FACS, wherein thedetection can be accomplished using radioactive, colorometric, orfluorescent labeling. One preferred method for measuring the amount ofABC1 protein in a cell sample is immunoprecipitation, whereinbiotinylated ABC1 proteins are contacted with anti-ABC1 antibody and thebound anti-ABC1 antibody is detected using streptavidin horse radishperoxidase.

The amount ABC1 protein in the test cell sample is compared with apre-determined standard amount of ABC1 protein. A pre-determinedstandard amount of ABC1 protein can be obtained by determining theaverage concentration of ABC1 protein found in cell samples taken from apopulation of mammalian subjects, wherein the mammalian subjects are thesame species as the subject from which the test cell sample wasobtained, and wherein the mammalian subjects do not have coronary heartdisease, Tangier disease, or other disease associated with lowHDL-cholesterol and are considered to have cholesterol efflux activitywithin a normal range (i.e., as indicated by an HDL-cholesterol levelwithin a normal range).

ABC1 Antibodies

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to protein. Fab and F(ab′)2 fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody(Wahl et al., J. Nucl. Med., 24:316-325 (1983)). Thus, these fragmentsare preferred, as well as the products of a FAB or other immunoglobulinexpression library. Moreover, antibodies of the present inventioninclude chimeric, single chain, and humanized antibodies.

Additional embodiments include chimeric antibodies, e.g., humanizedversions of murine monoclonal antibodies. Such humanized antibodies maybe prepared by known techniques, and offer the advantage of reducedimmunogenicity when the antibodies are administered to humans. In oneembodiment, a humanized monoclonal antibody comprises the variableregion of a murine antibody (or just the antigen binding site thereof)and a constant region derived from a human antibody. Alternatively, ahumanized antibody fragment may comprise the antigen binding site of amurine monoclonal antibody and a variable region fragment (lacking theantigen-binding site) derived from a human antibody. Procedures for theproduction of chimeric and further engineered monoclonal antibodiesinclude those described in Riechmann et al. (Nature 332:323, 1988), Liuet al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934,1989), and Winter and Harris (TIPS 14:139, May, 1993).

One method for producing an antibody comprises immunizing a non-humananimal, such as a transgenic mouse, with a polypeptide translated from apolynucleotide comprising SEQ ID NO: 1, a polynucleotide comprisingnucleotides 291-7074 of SEQ ID NO: 1, or a polynucleotide comprising anucleotide sequence that has at least 90% identity with a polynucleotidecomprising SEQ ID NO: 1, whereby antibodies directed against thepolypeptide translated from the described polynucleotides are generatedin said animal. Procedures have been developed for generating humanantibodies in non-human animals. The antibodies may be partially human,or preferably completely human. Non-human animals (such as transgenicmice) into which genetic material encoding one or more humanimmunoglobulin chains has been introduced may be employed. Suchtransgenic mice may be genetically altered in a variety of ways. Thegenetic manipulation may result in human immunoglobulin polypeptidechains replacing endogenous immunoglobulin chains in at least some(preferably virtually all) antibodies produced by the animal uponimmunization. Antibodies produced by immunizing transgenic animals witha polypeptide translated from any of the described polynucleotides areprovided herein.

Mice in which one or more endogenous immunoglobulin genes areinactivated by various means have been prepared. Human immunoglobulingenes have been introduced into the mice to replace the inactivatedmouse genes. Antibodies produced in the animals incorporate humanimmunoglobulin polypeptide chains encoded by the human genetic materialintroduced into the animal. Examples of techniques for production anduse of such transgenic animals are described in U.S. Pat. Nos.5,814,318, 5,569,825, and 5,545,806, which are incorporated by referenceherein.

Monoclonal antibodies may be produced by conventional procedures, e.g.,by immortalizing spleen cells harvested from the transgenic animal aftercompletion of the immunization schedule. The spleen cells may be fusedwith myeloma cells to produce hybridomas, by conventional procedures.

A method for producing a hybridoma cell line comprises immunizing such atransgenic animal with a immunogen comprising at least seven contiguousamino acid residues of a polypeptide translated from one of thedescribed polynucleotides; harvesting spleen cells from the immunizedanimal; fusing the harvested spleen cells to a myeloma cell line,thereby generating hybridoma cells; and identifying a hybridoma cellline that produces a monoclonal antibody that binds a polypeptidetranslated from one of the described polynucleotides. Such hybridomacell lines, and monoclonal antibodies produced therefrom, areencompassed by the present invention. Monoclonal antibodies secreted bythe hybridoma cell line are purified by conventional techniques.

The antibodies, upon specific binding to an ABC1 polypeptide, mayinhibit the activity of the ABC1 polypeptide. Preferably, the antibody,upon binding, inhibits the cholesterol transport activity of the ABC1polypeptide. Such antibodies can be made by immunizing a non-humananimal with a polypeptide corresponding to the region essential forcholesterol transport. The antibody can be tested to determine whetherit inhibits cholesterol efflux using any of the described cholesterolefflux assays. Such inactivating antibodies can be employed in an invitro assay, such as any of the cholesterol efflux assays describedherein, to determine whether a test compound promotes ABC1-mediatedcholesterol efflux. The inactivating antibodies can also be used in invitro assays to detect the comparative level of ABC1 protein in thecells of a mammalian subject. The inactivating antibodies are alsouseful in kits suitable for screening a compound to determine whetherthe compound modulates ABC1-dependent cholesterol efflux.

Kits for Identifying Therapeutic Compounds

The present invention also includes a kit suitable for screening acompound to determine the ABC1 expression modulating activity of acompound. The kit includes, in an amount sufficient to perform at leastone assay, a recombinant reporter construct comprising a reporter cDNAoperatively linked to an expression modulating portion of the mammalianABC1 gene, as a separately packaged reagent. Instructions for use of thepackaged reagent(s) are also typically included. The expressionmodulating portion of the mammalian ABC1 gene comprises the 5′ flankingsequence. In one preferred embodiment, the expression modulating portionof the mammalian ABC1 gene comprises SEQ ID NO: 3. In other preferredembodiments, the expression modulating portion of the mammalian ABC1gene comprises nucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643,1394-1643, or 1394-1532 of SEQ ID NO: 3. The reporter cDNA can be anysuitable reporter gene, including In a particularly preferredembodiment, the recombinant reporter construct is pAPR1. In anotherembodiment, the kit further comprises means for detecting the reporterprotein. Thus, the kit comprises reagents, such as buffers andsubstrates, used for reporter protein detection.

As used herein, the term “package” refers to a solid matrix or materialsuch as glass, plastic (e.g., polyethylene, polypropylene orpolycarbonate), paper, foil and the like capable of holding within fixedlimits a recombinant vector of the present invention. Thus, for example,a package can be a glass vial used to contain milligram quantities of acontemplated vector.

“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent—sample admixtures,temperature, buffer conditions and the like.

In addition, the present invention also includes a kit suitable forscreening a compound to determine whether the compound modulatesABC1-dependent cholesterol efflux.

In one embodiment, the kit includes, in an amount sufficient to performat least one assay, an inactivating anti-ABC1 antibody, as a separatelypackaged reagent. Instructions for use of the packaged reagent(s) arealso typically included.

In another embodiment, the kit includes an antisense ABC1oligonucleotide in an amount sufficient for at least one assay andinstructions for use. Preferably, the antisense ABC1 oligonucleotidecomprises SEQ ID NO: 53.

Microarrays

It will be appreciated that DNA microarray technology can be utilized inaccordance with the present invention. DNA microarrays are miniature,high-density arrays of nucleic acids positioned on a solid support, suchas glass. Each cell or element within the array contains numerous copiesof a single nucleic acid species that acts as a target for hybridizationwith a complementary nucleic acid sequence (e.g., mRNA). In expressionprofiling using DNA microarray technology, mRNA is first extracted froma cell or tissue sample and then converted enzymatically tofluorescently labeled cDNA. This material is hybridized to themicroarray and unbound cDNA is removed by washing. The expression ofdiscrete genes represented on the array is then visualized byquantitating the amount of labeled cDNA that is specifically bound toeach target nucleic acid molecule. In this way, the expression ofthousands of genes can be quantitated in a high throughput, parallelmanner from a single sample of biological material.

This high throughput expression profiling has a broad range ofapplications with respect to the ABC1 molecules of the invention,including, but not limited to: the identification and validation of ABC1disease-related genes as targets for therapeutics; molecular toxicologyof related ABC1 molecules and inhibitors thereof, stratification ofpopulations and generation of surrogate markers for clinical trials; andenhancing related ABC1 polypeptide small molecule drug discovery byaiding in the identification of selective compounds in high throughputscreens.

As discussed herein at Example 2, a method has been developed that usessamples of RNA derived from cells of an individual with a geneticabnormality and compares them to the RNA from a normal individual.Historically, identification of the cause of inherited diseases resultedfrom years of biochemical analysis or, more recently, from years of genemapping and positional cloning to identify the suspect gene within aninterval of millions of base pairs which had been shown to be closelylinked to the defect in inheritance studies (linkage analysis). The useof multigene expression analysis, most notably via “gene chips” canrevolutionize the pace of such discovery. Comparing the expression ofsamples of RNA derived from cells from an abnormal individual with agenetic disease versus RNA from an normal individual can quickly revealgenes whose corresponding mRNA is missing, severely underrepresented orseverely overrepresented in the abnormal diseased cell.

The term “individual” and used herein refers to any living organism thathas RNA such as, for example, mammals, plants. This method is preferablyused to identify the source of human genetic abnormalities. Morepreferably, the method is useful for detecting genetic sources of humancardiac and cardiovascular disorders such as identifying ABC1 as thegenetic defect in Tangier's disease.

The term “abnormality” as it is used herein refers to geneticdifferences that cause a physiological deviation in small number ofindividuals in a species in comparison to the majority of individuals ofthe species. The abnormality may be a positive abnormality or a negativeabnormality. For example, a positive plant abnormality would be agenetic difference that causes some individual plants to be droughtresistant in comparison to the other individuals in the species. Anegative abnormality would be one that causes an individual in the samespecies of plant to me more prone to drought damage than a normal plant.

The method can best be described by reference to our investigation intothe genetic cause of Tangier's disease. We began our investigation byusing RNA from cells cultured from an individual with Tangier disease toprobe microarrays containing nearly 60,000 normal human genes, and wewere able to use the probe results to identify ABC1 as the defectivegene in this monogenic disease in which patients have near zero levelsof circulating high density lipoprotein (HDL) and an increased risk ofheart disease. It is not necessary that the defective gene results in azero level of detectable mRNA signal in such an experiment. In thissuccessful example, roughly 175 out of the 58,800 probes on themicro-array were more than 2.5 fold underexpressed in the Tangierdisease RNA versus normal. Several additional steps may be taken toconfirm the identity of the culprit gene. They include repeating such amicro-array probe with an unrelated individual with Tangier disease,determining the chromosome map location of each gene to compare with areported large genetic interval that was linked to the disease,consideration of the likely function of the candidate proteins and theirhomologs, biochemical tests, and sequencing the best candidate gene inpatients to find mutations. In these ways, gene expression micro-arrayanalysis can lead to the identification of inherited genetic defectssuch as the identification of ABC1 as the defect in Tangier disease

A further utility in this method is that other genes that are eitherunder- or over-expressed in the disease sample vs. normal should includethose that are differentially regulated in consequence of the geneticdefect in the patient, either as compensatory responses or ascontributors to the disease pathology. This could provide identificationof other proteins in the relevant biological pathways that may beamenable to drug development and help elucidate the pathology of thedisease, with implications for treatment and diagnosis. In the caseswhere a gene deletion or other mutation causes complete absence of mRNA,as observed in many examples of thalassemia (globin gene defects) andother genetic diseases, gene expression analysis of disease versusnormal samples can lead to the identification of the missing gene in amore straightforward manner.

Although in these examples, the gene expression array that was probedwith RNA samples was of the type in which probe samples were cDNAsarrayed on microscope slides, alternative array technologies wouldsuffice. These would include, but not limited to those which array DNAsamples on filter membranes or use oligonucleotide probes synthesized on“gene chips” by photolithography.

Generally, the term microarray refers to an array of distinctoligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support. Microarrays may be prepared, used, and analyzed usingmethods known in the art. (See, e.g., Brennan, T. M. et al. (1995) U.S.Pat. No. 5,474,796; PCT application WO95/251116; Shalon, D. et al.(1995) PCT application WO95/35505; and U.S. Pat. No. 5,605,662 thespecifications of each of which are incorporated herein by reference)

A chemical coupling procedure and an ink jet device can be used tosynthesize array elements on the surface of the substrate. An arrayanalogous to a dot or slot blot may also be used to arrange and linkelements to the surface of a substrate using thermal, UV, chemical, ormechanical bonding procedures. A typical array may be produced by handor using available methods and machines and contain any appropriatenumber of elements. After hybridization, nonhybridized probes areremoved and a scanner used to determine the levels and patterns offluorescence. The degree of complimentrity and the relative abundance ofeach probe which hybridizes to an element on the microarray may beassessed through analysis of the scanned images.

Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereofmay comprise the elements of the microarray. Fragments suitable forhybridization can be selected using software well known in the art suchas LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragmentsthereof corresponding to the nucleotide sequences of an abnormalindividual and a normal individual are arranged on an appropriatesubstrate, e.g., a glass slide. The cDNA is fixed to the slide using,e.g., UV cross-linking followed by thermal and chemical treatments andsubsequent drying. (See, e.g., Schena, M. et al. (1995) Science270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645.) Probes,such as fluorescent probes are prepared and used for hybridization tothe elements on the substrate.

In order to conduct sample analysis using the microarrays, the RNA orDNA from a biological sample is made into hybridization probes. The mRNAis isolated, and cDNA is produced and used as a template to makeantisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies on the sequences, mutations, variants, or polymorphisms amongsamples.

The microarray is preferably composed of a large number of unique,single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs fixed to a solidsupport. Microarrays may contain oligonucleotides which cover the known5′, or 3′, sequence, or contain sequential oligonucleotides which coverthe full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5′ or more preferably at the 3′ end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. The oligomers are synthesized atdesignated areas on a substrate using a light-directed chemical process.The substrate may be paper, nylon or other type of membrane, filter,chip, glass slide or any other suitable solid support. An array may beproduced by hand or using available devises (slot blot or dot blotapparatus) materials and machines (including robotic instruments) andcontain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144dots, or any other multiple which lends itself to the efficient use ofcommercially available instrumentation.

Once the genetic causes of the inherited abnormality are narrowed oridentified, the potentially or actual defective portions of the genescan be used as targets in a microarray. The microarray can be used tomonitor the expression level of a large number of genes to develop andmonitor the activities of potential therapies and therapeutic agents.

The following examples further illustrate the present invention butshould not be construed to limit the present invention in any way.

Example 1

This example demonstrates that patients with Tangier Disease (TD) havean absence of apo A-I-mediated lipid efflux.

Cell Cultures: Human fibroblasts were obtained from skin explants fromtwo normal subjects (NL1) and three unrelated patients with Tangierdisease (TD). TD1 cells were obtained from a 53 year-old female withextremely low plasma HDL cholesterol and apo A-I levels and clinicalsymptoms typical of Tangier disease. TD2 cells were obtained from a 56year-old male with clinical, morphological, and biochemical features ofTangier disease, including very low levels of plasma HDL cholesterol andapoA-I (Francis, et al., J. Clin. Invest., 96, 78-87 (1995)). TD3 cellswere obtained from an 18 year-old male with Tangier disease whopresented with orange tonsil remnants, asymmetrical motor neuropathy,plasma HDL cholesterol of 5 mg/dL, and LDL cholesterol of 16 mg/dL (Lawnet al., J. Clin. Invest., 104, R25-R31 (1999)). The normal cells and TDsubject cells were immortalized as described in Oram et al., Lipid Res.,40: 1769-1781 (1999). Briefly, the cells were transfected withamphotropic retroviruses containing vectors with inserts of humanpapillomavirus 16 oncogenes E6 and E7 and a neomycin resistanceselectable marker. Control cells were infected with vector alone(mock-infected). Pooled cell populations were selected in the presenceof G418 for two passages, after which G418 was excluded from the medium.Fibroblasts were used between the fifth and sixteenth passage (primary)or sixth and fourteenth passage (immortalized). The immortalized normaland TD cells were seeded into 16-mm wells or 35-mm dishes and grown toconfluence in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetalbovine serum (FBS) before experimental use. RAW 264.7 mouse monocyticcells (American Type Culture Collection, Rockville, Md.) were alsomaintained in DMEM containing 10% FBS.

Assay to Measure Lipid Effux: Apo AI-mediated efflux of cholesterol andphospholipid was assayed according to the method described in Francis,et al., J. Clin. Invest., 96: 78-87 (1995). The cultured skinfibroblasts from normal and TD subjects were labeled by growth toconfluence in the presence of 0.2-0.5 μCi/ml [³H]cholesterol (40-60Ci/mmol, Amersham Corp., Arlington Heights, Ill.). The radioactivecholesterol was added to serum-containing growth medium when the cellswere 60-80% confluent. After 3 days, the cells were washed twice withPBS/BSA and simultaneously growth-arrested and cholesterol-loaded tomaximize apolipoprotein-mediated lipid efflux. This was achieved byincubating the cells for 48 hours in serum-free DMEM plus 2 mg/ml fattyacid-free bovine serum albumin (DMEM/BSA) (Sigma Chemical Co., St.Louis, Mo.) and 30 μg/ml non-radioactive cholesterol. RAW 264.7 cellswere cholesterol-loaded through the scavenger receptor by 24-hourincubation with acetylated LDL as described in Smith et al., J. Biol.Chem., 271:30647-30655 (1996). Briefly, RAW 264.7 cells in 24-welldishes were cholesterol-loaded and labeled overnight in 0.5 ml of DMEMsupplemented with 50 μl/ml 1M glucose, 10 μl/ml 200 mM glutamine and 2%BSA and with 50 μg/ml acetylated low density lipoprotein (AcLDL) and[³H]-cholesterol which had been pre-incubated for 30 minutes at 37° C.with AcLDL to yield a final concentration of 0.33 μCi/ml[³H]-cholesterol. Cells were subsequently washed five times with PBScontaining 1% BSA and incubated overnight (16-18 hours) in DMEM/BSA toallow for equilibrium of cholesterol pools.

After equilibrium of cholesterol pools, cells were rinsed four timeswith PBS/BSA and incubated for one hour at 37° C. with DMEM/BSA beforethe efflux incubations. Efflux medium (DMEM/BSA) containing eitheralbumin alone (control), albumin plus HDL (40 μg protein/ml), or albuminplus apo A-I (10 μg/ml, Biodesign International, Kennebunk, Me.) wasadded and the cells were incubated for 4, 24, or 48 hours. Phospholipidswere labeled by including 10 μCi/ml [³H]choline (75-85 Ci/mmol, AmershamCorp.) in the DMEM/BSA overnight equilibrium medium. The radioactivityfound in the culture medium was measured by scintillation counting aftera 15-minute centrifugation at 12,000 g. The radioactivity in the cellswas measured by scintillation counting after solubilization in 0.5 ml of0.2M NaOH (Smith et al., J. Biol. Chem., 271:30647-30655 (1996)) orextraction in hexane:isopropanol (3:2 v/v) as described in Francis, etal., J. Clin. Invest., 96, 78-87 (1995). Cells containing labeledphospholipids were extracted with 1 ml of isopropanol for 1 hour andthen with hexane:isopropanol as described above. The efflux ofcholesterol or phospholipid was expressed as the percentage of tritiatedlipid counts in the medium over the total tritiated lipid countsrecovered from the cells and medium (cpm medium/cpm(medium+lysate)×100).

As shown in FIGS. 1A and C, the addition of HDL or apo A-I results inthe removal of cholesterol from cholesterol-laden fibroblasts obtainedfrom normal subjects. However, in TD cells, the ability of HDL to removecholesterol is slightly diminished and the ability of apo A-I to removecholesterol is completely absent. FIG. 1 shows that normal and TDfibroblast cells release about 3-4% of the cellular [³H]-cholesterolinto the medium during 48-hour incubation with albumin. Addition of HDLto the albumin medium increased the efflux of [³H]-cholesterol from bothnormal and TD fibroblasts, although to a lesser extent with TD cells(FIGS. 1B, D). Addition of apo A-I promoted the efflux of[³H]-cholesterol from normal fibroblasts (FIGS. 1A, C), but had littleor no effect on [³H]-cholesterol efflux from TD fibroblasts (FIGS. 1B,D).

Example 2

This example demonstrates that 175 genes show at least 2.5-folddecreased expression and 375 genes show at least 2.5-fold increasedexpression in TD cells compared with normal cells. The differential geneexpression was determined using gene-expression microarray (GEM)analysis of cDNAs from normal individuals (non-TD) and from patientswith TD.

Cell Cultures: The immortalized cell cultures obtained from normalindividuals and TD patients described in Example 1 were used. Confluentcultures were maintained in DMEM/BSA and supplemented for 24 hours with1 mM 8-Bromo cyclic adenosine monophosphate (8-Br-cAMP, Sigma ChemicalCo., St. Louis, Mo.).

mRNA Extraction and cDNA Synthesis: mRNA from both normal and TDfibroblast cells was prepared from total RNA extracted from cells withTrizol (Life Technologies Inc., Bethesda, Md., Cat. #15596-026). ThemRNA was isolated using the Oligotex mRNA kit (Qiagen Inc., Valencia,Calif., Cat. #70022) according to vendor's protocols. The mRNA wasreverse transcribed using Cy3 or Cy5 fluorescent dye to createfluorescently labeled cDNA according to the method described in DeRisiet al., Science, 24:680-686 (1997). The resultant cDNA from TD cells waslabeled with Cy3 fluorescent dye and cDNA from normal cells was labeledwith Cy5 fluorescent dye (Incyte Genomics, Palo Alto, Calif.).

Microarray Analysis: To analyze differential gene expression in cellsfrom individuals with TD and normal individuals, Cy3 and Cy5 fluorescentlabeled cDNA samples prepared as described above were hybridized to aset of Gene Album microarrays (GEMs) on microscope slides (IncyteGenomics, Palo Alto, Calif.). Each of six slides contained about 9,800human cDNA samples plus 200 control samples, resulting in a microarrayof 58,800 partial cDNAs. Therefore, allowing for estimates ofredundancy, approximately 30-50% of expressed human genes wererepresented. The hybridization of Cy3-labeled cDNA prepared from TD1cells and Cy5-labled cDNA from normal cells allowed comparison of therelative RNA content of TD cells versus normal cells for the expressedgenes. In addition, Cy3-labeled cDNA prepared from TD2 cells andCy5-labled cDNA from normal cells were hybridized to the same set ofmicroarrays to examine the variation of gene expression betweendifferent TD patients.

Results: Data were analyzed using GemTools software (Incyte Genomics,Palo Alto, Calif.) and expressed as ratios of TD cell to normal cellmRNA. The results indicated that the majority of genes are comparablyexpressed in TD1 and normal cells. As shown in FIG. 2 (in the sectionabove and to the left of the diagonal) only 175 genes were more than2.5-fold underexpressed in TD1 cells compared with normal cells, whereas375 genes were more than 2.5-fold overexpressed in TD1 cells comparedwith normal cells (below and to the right of the diagonal). Genes morehighly expressed in the TD cells could include those that aredifferentially regulated as a consequence of the Tangier mutation,either as a compensatory response or as a contributor to the diseasepathology. Among the genes that could contribute to the observedphenotype of TD and are more highly expressed in TD cells includeinterferon-β (IFN-β), macrophage inflammatory protein-2α, granulocytechemotactic protein-2, IL-11, prostaglandin endoperoxide synthase-2(COX-2), thrombospondin, and monocyte chemotactic proteins 1, 3, and 4(Lawn et al., J. Clin. Invest., 104:R25-R31 (1999).

No single RNA that was expressed in the normal fibroblasts was foundcompletely absent in either the TD1 or TD2 cells. Also, comparison ofthe differentially expressed genes in TD1 and TD2 revealed very littlevariation between the individual TD patients. For instance, of the mosthighly down-regulated genes in TD2 cells, 92% were also under-expressedin TD1 cells compared with normal cells. One of the genes more than2.5-fold under-expressed in TD1 or TD2 versus normal cells was the genefor ABC1 protein. The ABC1 gene was pursued due to the ascribedfunctions of some of its homologues and also because the gene waslocalized to the approximate chromosome region reported as the TD generegion.

Example 3

This example demonstrates that the ABC1 gene is localized to the humanchromosome 9q31.

Previous genetic linkage analysis mapped the TD gene to the 7-cM regionof human chromosome 9q31 (Rust et al., Nat. Genet., 20, 96-98 (1998)).In addition, in situ hybridization analyses revealed that the ABC1 genewas localized to the broader chromosomal interval 9q22-9q31 (Luciani etal., Genomics, 21, 150-159 (1994)). Using PCR methods with theGeneBridge 4 panel of human/hampster radiation hybrids (ResearchGenetics, Inc., Huntsville, Ala.), human ABC1 was determined to belocated between the markers WI-14706 and WI-4062, corresponding to the7-cM region of human chromosome 9q31. DNA from 93 human/hampster hybridcell lines was amplified by PCR using human ABC1-specific primers LF:CCTCTCATTACACAAAAACCAGAC (SEQ ID NO: 11) and LR: GCTTTCTTTCACTTCTCATCCTG(SEQ ID NO: 12). Each line was scored as positive or negative for thehuman ABC1 amplification product and the mapping of ABC1 derived fromanalysis of this data was accomplished using the Whitehead Institute/MITCenter for Genome Research software, accessed via the internet. Theseresults were further confirmed by southern blot hybridization to humangenomic/yeast artificial chromosome clones (Research Genetics, Inc.)from the equivalent interval. In addition, public database searching(GeneMap '98; National Center for Biotechnology Information) andradiation hybrid mapping eliminated the other significantlyunderexpressed genes in the microarray data from the location in thereported genetic interval. These complementary data demonstrate that theABC1 gene is located on human chromosome 9q31 and further indicate thatthe ABC1 gene is associated with Tangier disease.

Example 4

This example shows the determination of the nucleotide sequence of thewildtype ABC1 gene, including the flanking regions and the entire codingregion.

DNA sequencing was performed using an ABI Prism 310 Genetic Analyzer orby Davis Sequencing (Davis, Calif.). Both strands were sequencedthroughout. The sequence of the open reading frame of the ABC1 gene froma normal subject was determined from a full-length cDNA clone obtainedfrom an expression plasmid library constructed from normal fibroblastRNA. To construct the plasmid library, cDNA was synthesized according tothe Stratagene kit protocol (Stratagene, La Jolla, Calif.). Briefly,first strand cDNA was synthesized from mRNA using an oligo-dT primerwith an XhoI site and MMLV reverse transcriptase in the presence of5-methyl dCTP. The second strand was synthesized using RNase H and DNApolymerase I in the presence of unmodified dNTPs. After the cDNA wasblunt-ended with pfu DNA polymerase, an EcoRI linker was ligated to thecDNA. The cDNA was then digested with XhoI, creating XhoI ends at the 3′end of the cDNA. The internal XhoI sites were protected from thisdigestion due to the semi-methylation during the first strand synthesis.The synthesized cDNA was cloned into the HindIII and XhoI sites of theplasmid pCEP4 (Invitrogen Corp., Carlsbad, Calif. #VO44-50), anexpression vector containing the cytomegalovirus promoter/enhancer. A585 bp ABC1 probe was generated by reverse transcriptase polymerasechain reaction (RT-PCR) using primers based on known ABC1 sequence,which were 5′-TCCTTGGGTTCAGGGGATTATC (SEQ ID NO: 13) and5′-CAATGTTTTTGTGGCTTCGGC (SEQ ID NO: 14). Using this ABC1 probe, a clonecontaining a 10.5 kb insert of human ABC1 cDNA was recovered from thelibrary using the CloneCapture selection kit according to themanufacturer's protocol (CLONTECH Laboratories, Inc., Palo Alto,Calif.). This clone is shown in FIG. 3 as pCEPhABC1. The 10.5 kb ABC1cDNA insert sequence is shown in SEQ ID NO: 1. Sequence determinationconfirmed that pCEPhABC1 contains the human ABC1 open reading frame of6783 nucleotides plus 5′ and 3′ untranslated regions, having a largeropen reading frame than the cDNA sequence reported by Langmann et al. inBiochem. Biophys. Res. Comm., 257, 29-33 (1999) (GenBank Accession No.AJO12376).

Example 5

This example demonstrates the sequence differences between the wildtypeABC1 gene and the TD1, TD2, and TD3 gene sequences.

cDNA Synthesis of TD1 TD2 and TD3: cDNA was prepared from TD1, TD2, andTD3 cells by reverse transcription polymerase chain reaction (RT-PCR)using the Superscript Choice cDNA system and the Advantage cDNApolymerase mix following the manufacturer's protocol (CLONTECH, PaloAlto, Calif.; Cat. #8417-1) using two sets of primer pairs designed fromthe normal human ABC1 gene sequence, designated: (1) sacIhabcf,5′-AGTCGAGCTCCAAACATGTCAGCTGTTACTGGAAGTGGCC (SEQ ID NO: 15); habcr3851,5′-TCTCTGGATTCTGGGTCTATGTCAG (SEQ ID NO: 16) and (2) habcf3585,5′-GGGAGCCTTTGTGGAACTCTTTC (SEQ ID NO: 17); habcrsalI,5′-ACTGGTCGACCATTGAATTGCATTGCATTGAATAGTATCAG (SEQ ID NO: 18).Amplification of 0.2-0.5 μg polyA+ RNA with these primers at a finalconcentration of 0.4 μM generated two overlapping templates ofapproximately 3.5 kb. The templates were gel-purified using the QIAEX IIsystem (QIAGEN, Inc., Valencia, Calif.; Cat. #20021) and adjusted to aconcentration of 100 ng/μl.

Sequencing of TD1 TD2 and TD3 cDNA: Eight μl of each template generatedas described above was sequenced in a reaction with individualsequencing primers designed on the basis of wildtype ABC1 sequence at afinal concentration of 0.5 μM. The primers were as follows:

1F: 5′TTTCCTGGTGGACAATGAA, (SEQ ID NO: 19) 2F: 5′-AGTGACATGCGACAGGAG;(SEQ ID NO: 20) 3F: 5′-GATCTGGAAGGCATGTGG; (SEQ ID NO: 21) 4F:5′-CCAGGCAGCATTGAGCTG; (SEQ ID NO: 22) 5F: 5′-GGCCTGGACAACAGCATA; (SEQID NO: 23) 6F: 5′-GGACAACCTGTTTGAGAGT; (SEQ ID NO: 24) 7F:5′-AAGACGACCACCATGTCA; (SEQ ID NO: 25) 8F: 5′-ATATGGGAGCTGCTGCTG; (SEQID NO: 26) 9F: 5′-GGGCATGAGCTGACCTATGTGCTG; (SEQ ID NO: 27) 10F:5′-AAGAGACTGCTAATTGCC; (SEQ ID NO: 28) 11F: 5′-AGCGACAAAATCAAGAAG; (SEQID NO: 29) 12F: 5′-TGGCATGCAATCAGCTCT; (SEQ ID NO: 30) 13F:5′-TCCTCCACCAATCTGCCT; (SEQ ID NO: 31) 14F: 5′-TTCTTCCTCATTACTGTT; (SEQID NO: 32) 15F: 5′-GATGCCATCACAGAGCTG; (SEQ ID NO: 33) 16F:5′-AGTGTCCAGCATCTAAA; (SEQ ID NO: 34) 1R: 5′-CAAAGTTCACAAATACTT; (SEQ IDNO: 35) 2R: 5′-CTTAGGGCACAATTCCACA; (SEQ ID NO: 36) 3R:5′-TGAAAGTTGATGATTTTC; (SEQ ID NO: 37) 4R: 5′-TTTTTCACCATGTCGATGA; SEQID NO: 38) 5R: 5′-CTCCACTGATGAACTGC; (SEQ ID NO: 39) 6R:5′-GTTTCTTCATTTGTTTGA; (SEQ ID NO: 40) 7R: 5′-AGGGCGTGTCTGGGATTG; (SEQID NO: 41) 8R: 5′-CAGAATCATTTGGATCAG; (SEQ ID NO: 42) 9R:5′-CATCAGAACTGCTCTGAG; (SEQ ID NO: 43) 10R: 5′-AGCTGGCTTGTTTTGCTTT, SEQID NO: 44) 11R: 5′-TGGACACGCCCAGCTTCA, (SEQ ID NO: 45) 12R:5′-CCTGCCATGCCACACACA, (SEQ ID NO: 46) 13R: 5′-CTCATCACCCGCAGAAAG, (SEQID NO: 47) 14R: 5′-CACACTCCATGAAGCGAG, (SEQ ID NO: 48) 15R:5′-TCCAGATAATGCGGGAAA, (SEQ ID NO: 49) 16R: 5′-TCAGGATTGGCTTCAGGA, (SEQID NO: 50) UTR1R: 5′-AAGTTTGAGCTGGATTTCTTG. (SEQ ID NO: 51)

Results: The nucleotide numbering follows the numbering found in Lawn etal. (1999). Patient TD1 retained the full open reading frame, with 2substantial differences from the wild-type sequence (SEQ ID NO: 8). Oneof these is an A to G substitution, resulting in a change from aglutamine to arginine residue at position 537 of the 2201 amino acidsequence, as published by Lawn et al. (1999). The location of thisresidue is within the NH2-terminal hydrophilic domain, near the firstpredicted transmembrane domain. Patient TD2 also retained the openreading frame with an arginine to tryptophan substitution at residue 527(SEQ ID NO: 10). Thus, both TD1 and TD2 contain a substitution alteringthe charge of an amino acid in the same region of the protein. TD3 DNAcontains a 14 nucleotide insertion in its ABC1 cDNA following nucleotide5697 in one allele and a 138 bp insertion after nucleotide 5062 in theother allele.

Genomic sequencing of the TD1, TD2, and TD3 DNAs confirmed the changesfound in the respective cDNAs. The genomic sequence was generated by PCRamplification of a 156 bp region of genomic DNA isolated fromfibroblasts that contained the mutations found in the cDNA from TD1 andTD2. The genomic sequencing also indicated that patient TD1 washomozygous for the glutamine to arginine substitution. Genomic DNAanalysis showed that TD2 was a compound heterozygote with one allelecontaining the detected substitution and the second allele (which failedto produce detectable mRNA) containing an undetermined defect. Neitherof the substitution mutations was found in more than 80 alleles ofgenomic DNA of non-TD individuals. TD3 insertions were identified bysequence analysis and confirmed by RT-PCR using primers surrounding theinsertion points. The 14-bp insertion following nucleotide 5697 causes aframeshift, resulting in the replacement of the wild-type amino acidsequence from a location before the second ATP binding domain, up to thepoint of a premature protein termination. The 138 bp insertion followingnucleotide 5062 in the other allele contains an inframe stop codon.

Example 6

This example demonstrates that inhibitors of ABC1 transport activityalso inhibit apo A-I-mediated cholesterol efflux from fibroblast cells.

To test whether inhibition of ABC1 could affect the process ofapolipoprotein-mediated cholesterol efflux, two compounds reported to beABC1 inhibitors were tested in assays which monitor apolipoproteinmediated cholesterol efflux. The compounds4,4-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) andsulphobromophthaleine (BSP) were reported to inhibit anion transportactivities of ABC1 in a dose-dependent fashion (Becq et al., J. Biol.Chem., 272:2695-2699 (1997); Hamon et al., Blood, 90:2911-2915 (1997)).The apolipoprotein-mediated cholesterol efflux assays were performed asdescribed in Example 1 with the noted changes. Cholesterol-loaded and[³H]cholesterol-labeled normal fibroblasts (n=3) were incubated for 6hours with or without 5 μg/ml apo A-I and either 0, 0.2 mM, or 0.4 mMDIDS. In addition, cholesterol-loaded and [³H]cholesterol-labeled normalfibroblasts (n=3) were incubated for 6 hours with or without 5 μg/ml apoA-I and either 0, 0.2 mM, or 0.4 mM BSP. [³H]cholesterol efflux wasmeasured by scintillation counting as described in Example 1 andcalculated as the percentage of total radiolabeled cholesterol appearingin the medium. The results are shown in FIG. 5 as the mean ±SD (n=3) ofefflux in the presence of apo A-I after subtraction of values for apoA-I-free medium. FIG. 5 shows that both DIDS and BSP inhibit the 6-hourefflux of tritiated cholesterol mediated by apolipoprotein A-I. Inaddition, similar inhibition was observed with the efflux of tritiatedphosphatidyl choline using DIDS and BSP (data not shown). The results ofthese tests mimic the efflux defect in fibroblasts derived from patientswith TD, described in Example 1.

Example 7

This example demonstrates that antisense inhibition of ABC1 mRNAexpression inhibits apo A-I-mediated cholesterol efflux from fibroblastcells.

Normal skin fibroblasts were labeled with [³H]cholesterol as describedin Example 1. The cells were then loaded with oligonucleotide byscraping in the presence of either 30 μM control Morpholinooligonucleotide (5′-CCTCTTACCTCAGTTACAATTTATA-3′ corresponding to theantisense complement of a β-globin thalassemic mRNA; SEQ ID NO: 52) or30 μM ABC1 antisense Morpholino oligonucleotide(5′-CATGTTGTTCATAGGGTGGGTAGCTC-3′; SEQ ID NO: 53) and reseeding on newdishes. Control cells were mock-loaded after [³H]cholesterol-labeling byscraping and reseeding in the absence of oligonucleotide. ApoA-I-mediated efflux was measured after 12 hours by scintillationcounting as the percentage of total radiolabeled cholesterol appearingin the medium. The results are shown in FIG. 6 as the mean ±SEM of threeseparate experiments, normalized to the value for apo A-I-specificefflux in the absence of oligonucleotide in each experiment. As shown inFIG. 6, antisense oligonucleotides directed against ABC1 mRNA caused a50% reduction in cholesterol efflux from normal fibroblasts comparedwith control antisense oligonucleotide (β-globin antisenseoligonucleotide).

Example 8

This example demonstrates that over expression of the human ABC1 generesults in an increase in apo A-I-mediated cholesterol efflux frommonocyte cells.

Stable Transfection of RAW 264.7 Cells: Mouse monocytic RAW 264.7 cellswere stably transfected with the pCEPhABC1 expression plasmid for humanABC1. Construction of the pCEPhABC1 plasmid containing the open readingframe of human ABC1 is described in Example 4. Approximately 1×10⁶ RAW264.7 cells were transfected for 5 hours with 2 μg of pCEPhABC1 DNA and12 μl Geneporter transfection reagent (Gene Therapy Systems, Inc., SanDiego, Calif.; Cat. #T201007) in 0.8 ml serum-free DMEM. Two days later,cells were split at ratios ranging from 1:2-1:50 and selection appliedby adding 150 μg/ml hygromycin to the culture medium. After two weeks,hygromycin-resistant colonies were picked and expanded.

Apo A-I-mediated Cholesterol Efflux Assay: Parental RAW 264.7 cells andthree clonal lines (L3, L5, and L6) stably expressing human ABC1 weregrown to confluence. The cells were cholesterol-loaded and labeled byincubation for 24 hours with 0.5 μCi/ml [³H]cholesterol and 50 μg/mlacetylated LDL as described in Example 1. After equilibrium ofcholesterol pools by an overnight incubation in DMEM/BSA, cells werewashed and the efflux medium was added as described in Example 1. ApoA-I-mediated cholesterol efflux was measured as previously described byscintillation counting of the tritiated cholesterol in the cell medium,expressed as a percentage of the total counts recovered from the cellsand medium. The results are presented as the mean ±SEM of three separateexperiments normalized to the value for apo A-I-specific efflux fromparental RAW 264.7 cells within each experiment. FIG. 7 shows the apoA-I-mediated cholesterol efflux from parental RAW 264.7 cells and L3,L5, and L6 transfected cell lines. As can be seen, transfection with theABC1 expression vector results in a 4-fold (L6) to 8-fold (L3 and L5)increase in apo A-I-mediated cholesterol efflux. These results indicatethat overexpression of the ABC1 gene can substantially increase theamount of cholesterol efflux from macrophage cells.

Example 9

This example demonstrates that ABC1 mRNA expression is regulated bycellular conditions related to cholesterol efflux in normal skinfibroblasts, but not in TD fibroblasts.

To determine whether ABC1 plays a rate-limiting role in cellular sterolefflux, the synthesis of ABC1 was measured under various cellularconditions related to cholesterol efflux processes. Specifically, normalfibroblasts and TD fibroblasts were individually exposed to conditionsof excess cAMP, cholesterol, or Apo A-I. Cell cultures of normal skinfibroblasts and TD1 and TD2 fibroblasts were prepared as described inExample 1. The level of ABC1 mRNA was measured by RT-PCR.

Cell cultures: Immortalized cell cultures of normal skin fibroblasts andTD1 and TD2 fibroblasts were prepared as described in Example 1. Cellswere grown to subconfluence in DMEM/10% FBS before replacement withDMEM/BSA and the indicated additive for 24 or 48 hours. RNA was preparedas described in Example 2.

RT-PCR: Quantitative PCR was carried out using the GeneAmp 5700 SequenceDetection System (Perkin-Elmer Applied Biosystems, Foster City, Calif.).Briefly, 500 ng of DNase-treated mRNA was reverse transcribed usingrandom hexamer primers at 2.5 μM. Approximately 5% of this reaction wasamplified by PCR using the SYBR green core kit (PE Applied Biosystems,Foster City, Calif.; Cat. #430-4886) and human ABC1 primers LF:5′-CCTCTCATTACACAAAAACCAGAC (SEQ ID NO: 11) and LR:5′-GCTTTCTTTCACTTCTCATCCTG (SEQ ID NO: 12) to yield an 82 bp fragmentcorresponding to nucleotides 7018-7099 of human ABC1. PCR cycleconditions were as follows: 10 minutes 95° C.; followed by 40 cycles of95° C., 15 seconds; and 60° C., 60 seconds. The mRNA in each sample wasquantitated by detecting the increase in fluorescence caused by SYBRgreen binding to the double-stranded amplification product generatedduring each PCR cycle. All samples were run in triplicate and normalizedagainst β-actin mRNA, amplified in parallel reactions with primersactinF: 5′-TCACCCACACTGTGCCATCTACGA (SEQ ID NO: 54) and actinB:5′-CAGCGGAACCGCTCATTGCCAATGG (SEQ ID NO: 55). Standard curves were runfor both ABC1 and β-actin on the same PCR plate.

8-Br-cAMP Assay: Normal, TD1, and TD2 fibroblast cells were grown tosubconfluence in DMEM/10% FBS and then treated with 1 mM 8-Br-cAMP inDMEM/BSA for 24 hours.

Cholesterol Assay: Normal, TD1, and TD2 fibroblast cells were grown tosubconfluence in DMEM/10% FBS and then treated with 30 μg/ml freecholesterol in DMEM/BSA for 48 hours followed by 18-24 hours ofequilibrium in DMEM/BSA.

Apo A-I Assay: Normal, TD1, and TD2 fibroblast cells were grown tosubconfluence in DMEM/10% FBS and then treated with 30 μg/ml freecholesterol in DMEM/BSA for 48 hours followed by 18-24 hours ofequilibrium in DMEM/BSA plus 10 μg/ml apo A-I.

Results: FIG. 8 shows that in normal fibroblasts ABC1 mRNA is increasedapproximately 10-fold by exposure to 8-Br-cAMP and increasedapproximately 17-fold by exposure to cholesterol in serum-free medium.Subsequent exposure of cholesterol-loaded cells to Apo A-I results in amarked decrease in ABC1 mRNA expression. Although the mechanism has notbeen demonstrated, previous studies have shown that cholesterol effluxis promoted in the presence of such compounds as cAMP and cholesterol(Hokland et al., J. Biol. Chem., 268:25343-25349 (1993)). The presentresults indicate that in normal fibroblasts, ABC1 mRNA is induced bythese known effectors of the cholesterol efflux pathway and repressed byexposure to an apolipoprotein cholesterol acceptor, demonstrating thatthe expression of ABC1 is regulated by cellular conditions related toapolipoprotein-mediated cholesterol efflux. In contrast, fibroblastcells from TD patients are not regulated by effectors of cholesterolefflux. First, the cAMP-inducible level of ABC1 mRNA in both TD1 and TD2cells is only approximately 40% of that in normal cells. Further,exposure of cholesterol-loaded cells to Apo A-I either did not alterABC1 expression (TD1 cells) or slightly increased ABC1 expression (TD2cells). These results reflect the defect in Apo-A-I-mediated cholesterolefflux described for TD cells. Interestingly, growth of cells inserum-containing medium suppressed ABC1 message to near the limit ofdetection (data not shown). This may reflect the fact that functioningof the lipid efflux pathway requires cell quiescence or other cellularstates of reduced cholesterol need. In conclusion, conditions that areassociated with increased efflux of cellular cholesterol (i.e.,cholesterol loading, cAMP treatment, serum deprivation) also result inincreased expression of ABC1 mRNA in normal fibroblast cells.Conversely, exposure of cholesterol-loaded normal fibroblast cells toapo A-I reduces ABC1 expression.

Example 10

This example demonstrates that ligands for the LXR nuclear receptor,such as 20-hydroxycholesterol, and ligands for the RXR receptor, such as9-cis retinoic acid, can increase ABC1 gene expression in mouse RAW264.7 cells.

LXR nuclear receptors are transcription factors that form obligateheterodimers with the nuclear receptor RXR, and are activated to enhancetranscription of their target genes by binding a class of oxysterolsincluding 22-hydroxycholesterol and 20-hydroxycholesterol (Janowski etal., Nature, 383:728-731 (1996)). As such, they are candidates for themediation of cholesterol-induced gene transcription. Further, in lightof studies which showed that ABC1 mRNA and protein increase infibroblasts and macrophages in response to cholesterol loading, andother studies which showed that LXR and RXR expression increase incholesterol-loaded macrophage cells by exposure to oxidized LDL, the LXRand RXR nuclear receptors are highly plausible candidates fortranscriptional activators of the ABC1 gene. To determine whether LXRand RXR receptors play a role in ABC1 gene expression, the level of ABC1mRNA was measured in response to 20-hydroxycholesterol and 9-cisretinoic acid.

Mouse RAW 264.7 cells were grown to subconfluence in DMEM/10% FBS andthen treated for 24 hours in serum-free DMEM/BSA with either 9-cisretinoic acid (10 μM), 20-hydroxycholesterol (10 μM), or both ligandstogether (20 μM total). Control cells received ethanol vehicle only(0.1% v/v). RNA was extracted, treated with DNase, and ABC1 mRNAmeasured by RT-PCR using PE Biosystems SYBR Green Technology asdescribed in Example 9. FIG. 9 shows that treatment with either20-hydroxycholesterol or 9-cis retinoic acid results in an increase inABC1 mRNA expression. In addition, FIG. 9 shows that treatment with bothligands together results in a markedly synergistic effect, with anapproximate 6-fold increase over the ABC1 expression observed witheither ligand alone. These results demonstrate that ligands for thenuclear receptors LXR and RXR can increase the expression of the ABC1gene.

Example 11

This example demonstrates that enhanced expression of ABC1 protein inthe plasma membrane is associated with lipid efflux.

Cell-surface labeling and immunoprecipitation was used to determinewhether increased expression of ABC1 protein in the plasma membrane iscorrelated with an increase in cholesterol efflux (FIG. 10). Therelative amount of ABC1 on the cell surface was determined bycross-linking surface proteins on intact cells with themembrane-impermeable agent sulfo-NHS-biotin, followed by the steps ofmembrane solubilization, immunoprecipitation with ABC1 antibody,SDS-PAGE, and detection with streptavidin.

Cell Culture: Normal and TD1 fibroblast cells were immortalized asdescribed in Example 1. Both normal and TD1 cells were cultured undercontrol conditions and conditions known to increaseapolipoprotein-mediated cholesterol efflux (Oram, et al., J. Lip. Res.,40: 1769-1781 (1999)). Control cells were grown to confluence inDMEM/10% FBS and then incubated in DMEM/BSA for 18 hours with noadditives (control). cAMP-treated cells were grown to confluence inDMEM/10% FBS and then incubated in DMEM/BSA for 18 hours with 1 mM8-Br-cAMP (cAMP). Cholesterol-loaded cells were grown to confluence inDMEM/10% FBS and then incubated in DMEM/BSA for 48 hours with 30 μg/mlcholesterol plus 18 hours with no additives (cholesterol).Cholesterol-loaded cells treated with cAMP were grown to confluence inDMEM/10% FBS and then incubated in DMEM/BSA for 48 hours with 30 μg/mlcholesterol plus 18 hours with 1 mM 8-Br-cAMP (cholesterol+cAMP).

Cell-Surface Labeling: For selective labeling of plasma membrane ABC1,the cells were incubated for 30 minutes at 0° C. with PBS containing 1mg/ml sulfo-NHS-biotin (Pierce, Rockford, Ill.; Cat. #21217) tobiotinylate cell-surface proteins (see Walker et al., Biochemistry,50:14009-14014 (1993)).

Immunoprecipitation: Rabbit antiserum for ABC1 was raised against asynthetic peptide corresponding to the deduced peptideKNQTVVDAVLTSFLQDEKVKES (SEQ ID NO: 60) located at the C-terminus ofhuman ABC1. Immunoprecipitation was performed by solubilizing the cellsin PBS containing 1% Triton X-100 (Sigma, St. Louis, Mo.) and proteaseinhibitors leupeptin (1 mM), pepstatin (1 mM), and aprotinin (1 mM). Thecell extract was incubated overnight at 4° C. with anti-ABC1 antiserumat 1:200 dilution followed by an additional 1 hour incubation with 5 μlproteinA-coated magnetic beads (Dynal, Lake Success, N.Y.; Cat.#1001.01). The antibody-antigen complex was sedimented with a magnet,the beads were washed twice with 1% Triton-X/PBS, and the proteins wereeluted with 1% acetic acid.

Detection of ABC1 Protein: The eluted biotinylated proteins weresubjected to SDS-PAGE (6% gel; 150V, 5 hours) and transferred tonitrocellulose membrane (200 mA, 18 hours). The nitrocellulose wasprobed with streptavidin-horse radish peroxidase (Amersham Pharmacia,Piscataway, N.J.; Cat. #RPN 1231) diluted 300-fold and detected byenhanced chemiluminescence labeling (ECL) according to vendor's protocol(Amersham Pharmacia, Piscataway, N.J.). To test for possiblebiotinylation of intracellular proteins, the post-immunoprecipitationsupernatant was treated with a mouse monoclonal antibody to theintracellular protein β-COP and immunoprecipitated biotinylated β-COPwas assayed by streptavidin blotting. None was detected.

Results: As shown in FIG. 10, the 240 kDa ABC1 protein appears as adoublet. The ABC1 protein is partially localized to the plasma membranein both normal (10A) and TD1 (10B) fibroblast cells. Similar resultswere seen with a second normal fibroblast cell line and with TD2fibroblasts (data not shown). Cell-surface expression of ABC1 wasincreased slightly when cells grown in serum (normal and TD1 cells) weretreated with 8-Br-cAMP. Serum deprivation and cholesterol-loading ofboth normal and TD1 cells markedly increased cell-surface expression ofABC1, which was further enhanced by cAMP treatment. These resultsindicate that expression of ABC1 at the cell surface is regulated byconditions that enhance apolipoprotein-mediated lipid efflux, consistentwith the idea that its localization to the plasma membrane plays a keyrole in its lipid transport function. The mutations in TD1 and TD2 cellsdo not appear to severely impair expression or processing of ABC1,implying that secondary effects on lipid transport or interactions withaccessory proteins depend on its NH2-terminal domain, where themutations occur.

Example 12

This example shows that agents that inhibit the degradation of 3′5′cyclic AMP, such as phosphodiesterase inhibitors, increaseapolipoprotein A-I-mediated efflux from macrophage cells.

As shown in FIG. 10, cAMP increases the activity of ABC1. The presentstudies were performed to determine the cholesterol efflux frommacrophage cells in the presence of elevated cAMP. Elevated levels ofcAMP can be attained in the presence of agents that either stimulatecAMP synthesis or inhibit the degradation of cAMP. For example, rolipramis a compound that regulates cAMP levels by inhibitingphosphodiesterases, a group of enzymes that degrade cAMP. The effect ofelevated cAMP on cholesterol efflux was determined using theapolipoprotein-mediated cholesterol efflux assays described inExample 1. Briefly, RAW 264.7 cells suspended at a density of 1.25×10⁵cells/ml were grown in DMEM/10% FBS supplemented with pyruvate. After 24hours, the medium was removed and replaced with DMEM/BSA plusradiolabeled cholesterol (1 μCi/ml ³ [H]-cholesterol) and 50 μg/ml ofacetylated LDL for 24 hours. The cells were then maintained for 24 hoursin equilibrium medium consisting of DMEM/BSA plus either apo A-I alone(20 μg/ml), apo A-I and 8-bromo 3′,5′ cAMP (1 mM) or apo A-I androlipram (50 μM). After 12-24 hours, [³H]cholesterol efflux was measuredby scintillation counting as described in Example 1 and calculated asthe percentage of total radiolabeled cholesterol appearing in themedium. The results indicated that cholesterol-loaded control cells thatreceived no apo A-I showed a 3% cholesterol efflux, while cells thatreceived apo A-I only showed a 5% efflux. Cholesterol-loaded cells thatreceived apo A-I and cAMP showed a 32% cholesterol efflux, demonstratingthat elevated cAMP promotes cholesterol efflux. Similarly, cells thatreceived apo A-I and a phosphodiesterase inhibitor (rolipram) showed a17% cholesterol efflux.

Example 13

This example shows that agents that are ligands for nuclear receptors,such as LXR, RXR, and PRAR nuclear receptors, increase apolipoproteinA-I-mediated efflux from macrophage cells.

To determine whether ligands for nuclear receptors affect the process ofapolipoprotein-mediated cholesterol efflux, various ligands were testedusing the apo A-I-mediated cholesterol efflux assay described in Example12. The nuclear receptor superfamily includes several members, such asthe liver receptor LXR, the retinoid receptor RXR, and the peroxisomeproliferator-activated receptor PPAR, which have been implicated inlipid metabolism (Russell. D. W., Cell, 97:539-542 (1999); Spiegelman,B. W., Cell, 93:153-155 (1998); Janowski et al., Nature, 383:728-731(1996)). Further, ligands for some of these receptors have been observedto increase plasma HDL and gene expression profiling (microarray) datahave shown that hormone receptors respond to cholesterol loading viaoxidized LDL. Using the above-described assay, 9 cis-retinoic acid (RXRligand), oxysterol (LXR ligand), and fenfibrate (PPAR ligand) was testedto determine the effect on cholesterol efflux. Cholesterol-loadedcontrol cells that received no apo A-I showed a 3% cholesterol efflux,while cells that receive apo A-I only showed a 5% efflux. In contrast,cholesterol-loaded cells that receive apo A-I and 9 cis-retinoic acid(30 ng/ml) showed a 16% cholesterol efflux. Cells that receive apo A-Iand oxysterol (5 μg/ml) showed a 14% cholesterol efflux. Cells thatreceive apo A-I and fenfibrate (3 μg/ml) showed a 10% cholesterolefflux. These results indicate that hormone receptors may be modulatedto increase the rate of apolipoprotein-mediated cholesterol efflux frommacrophages.

Further, when the efflux assay was performed using variousconcentrations of 9-cis-RA (0.3 ng/ml, 3.0 ng/ml, or 30 ng/ml), theresults showed that 9-cis-RA mediated cholesterol efflux from macrophagecells in a dose-dependent manner. Specifically, control cells (apo A-Ionly) showed 1890 c.p.m., 0.3 ng/ml 9-cis-RA showed 1522 c.p.m., 3.0ng/ml 9-cis-RA showed 3568c.p.m., and 30 ng/ml 9-cis-RA showed 8597c.p.m. In addition, using a similar assay where RAW 264.7 cells werecholesterol-loaded for 48 hours, other nuclear receptor activators, suchas 22-hydroxycholesterol (LXR ligand) and benzfibrate, were shown toincrease cholesterol efflux (data not shown).

Example 14

This example shows that eicosanoids, such as prostaglandin E1 andprostacyclin PG12, increase apolipoprotein A-I-mediated efflux frommacrophage cells.

Eicosanoids, such as prostaglandins and prostacyclins, have been shownto be effective in the treatment of hypercholesterolemia. To determinewhether eicosanoids affect the process of apolipoprotein-mediatedcholesterol efflux, PGE1 and PG12 were tested using the apo A-I-mediatedcholesterol efflux assay described in Example 12. This assay showed thatcholesterol-loaded control cells that receive no apo A-I have a 3%cholesterol efflux, while cells that receive apo A-I only have a 5%efflux. Cholesterol-loaded cells that receive apo A-I and PG12 (25 nm)showed a 10% cholesterol efflux. Cells that receive apo A-I and PGE1 (25nM) showed a 15% cholesterol efflux. These results demonstrate thateicoasnoids can increase the rate of apolipoprotein-mediated cholesterolefflux from macrophages.

Example 15

This example demonstrates that a reporter gene under the control of anABC1 promoter can be used to test compounds for the ability to regulateABC1 gene expression.

The pGL3 luciferase reporter vector system (Promega, Madison, Wis.) wasused to create a recombinant plasmid to measure reporter gene expressionunder control of the ABC1 promoter.

Construction of Reporter Plasmids: Plasmid pGL3-Basic (Promega, Madison,Wis.; Cat. #E1751) was used as a control plasmid containing thepromoterless luciferase gene. The reporter construct containing the ABC1promoter and luciferase gene was made by cloning a genomic fragment fromthe 5′ flanking region of the ABC1 gene (hAPR15′ promoter, correspondingto nucleotides 1080-1643 of SEQ ID NO: 3) into the SacI site of theGL3-Basic plasmid to generate plasmid GL-6a. Next, plasmid GL-6a wasdigested with SpeI and Acc65I. A BsiWI-SpeI fragment excised from alambda subclone, representing the ABC1 genomic sequence corresponding tonucleotides 1-1534 of SEQ ID NO: 3 was ligated into the remainingvector/ABC1 promoter fragment produced by this digestion. The resultantplasmid, pAPR1, encodes the luciferase reporter gene undertranscriptional control of 1.75 kb of the human ABC1 promoter sequence.

Transfection of Reporter Constructs: The above-described control orpAPR1 plasmid was transfected into confluent cultures of RAW 264.7 cellsmaintained in DMEM containing 10% fetal bovine serum. Each well of a 12well dish was transfected for 5 hours with either pGL3-Basic,pGL3-Promoter or pAPR1DNA (1 μg), luciferase plasmid DNA (1 μg), and 12μl of Geneporter reagent (Gene Therapy Systems, San Diego, Calif.; Cat.#T201007). In addition, 0.1 μg of pCMV, plasmid DNA (Clontech, PaloAlto, Calif., Cat. #6177-1) was added as a control for transfectionefficiency. After 5 hours, the culture medium was replaced withserum-free DMEM/BSA in the presence of or absence of acetylated LDL (100μg/ml) and incubated for 24 hours.

For added convenience in high throughput screening, cultured cells canbe stably transfected with reporter plasmids using the followingprocedure. First, 5×10⁶ RAW 264.7 cells are transfected for 5 hours in a60 mm dish with 9 μg of the pAPR1 plasmid and pCMVscript (Stratagene,LaJolla, Calif.) in 10 ml of serum-free DMEM with 50 μl Geneportertransfection reagent (Gene Therapy Systems, San Diego, Calif.).Subsequently, the transfection medium is replaced with complete mediumand the cells incubated overnight at 37° C. Subsequently, the cells aretransferred to separate dishes at dilutions ranging from 1:5 to 1:1000and incubated in selection medium containing 800 μg/ml G418 (LifeTechnologies, Bethesda, Md.) for 20 days. Visible colonies are picked,expanded, and assayed for luciferase activity as described below. Usingthis method, five clonal cell lines positive for luciferase activitywere identified for use in high throughput assays.

Luciferase Assay: Following transfection, the cells in each well werelysed in 70 μl of 1× cell lysis reagent (Promega, Madison, Wis., Cat.#E3971), subjected to one freeze-thaw cycle, and the lysate cleared bycentrifugation for 5 minutes at 12,000 g. After centrifugation, 100 μlof luciferase assay reagent (Promega, Madison, Wis.; Cat. #E1501) wasadded to 10 μl of lysate. The luciferase activity of each lysate wasmeasured as light units using a luminometer. Additionally, theβ-galactosidase activity of each lysate was measured using thechemiluminescent assay reagents supplied in the Galacto-light kitaccording to the manufacturer's instructions (Tropix Inc., Bedford,Mass.: Cat. #BL100G). The normalized luciferase activity for each lysatewas determined by dividing the luciferase activity value by thedetermined β-galactosidase value and reported as relative light units.

Results: The luciferase activity detected in cells transfected withpAPR1 was 3.3-fold higher than the activity detected in control cellstransfected with pGL3-Basic plasmid containing luciferase cDNA only.These results indicated that the transcriptional regulatory regions ofABC1 were in place. When the pAPR1 transfected cells were incubated with100 μg/ml acetyl LDL for 24 hours, the luciferase activity was 3.25-foldhigher than in cells that had not been treated with acetyl LDL. Theseresults suggest that the genomic ABC1 sequence contains a “cholesterolresponsive” element found in the 5′ flanking region which mediates thecholesterol loading response of the native ABC1 gene. This reportersystem can also be used to test other compounds to determine whether thecompound modulates ABC1 expression.

Example 16

This example demonstrates an additional assay that can be used to testcompounds for the ability to regulate ABC1 gene expression using areporter gene under the control of an endogenous ABC1 promoter.

This assay involves constructing a recombination vector that contains apromoterless reporter gene and a selection marker gene. The vector islinearized and transfected into cells such that the reporter gene isintegrated into the cellular genome downstream of the endogenous ABC1promoter. Using this assay, expression of the reporter gene is driven bythe endogenous ABC1 promoter in response to a test compound.

Construction of Reporter Plasmids: The recombination vector containing apromoterless reporter gene can be made starting with a 7 kb EcoRIgenomic fragment of ABC1 that contains exon 0 (which includes the ABC1start sites) and part of intronl. Using site-directed mutagenesis, aSalI restriction site can be generated in the exon 0 sequence downstreamof the two known start sites. The recombination vector is generated byinserting a DNA fragment containing a promoterless reporter gene, suchas luciferase, and a promoterless selective marker, such as puromycinresistance, into the SalI site. An internal ribosome entry signal shouldbe inserted between the reporter gene and marker gene so that the geneswill be transcribed in the correct orientation. The recombination vectorcontains two Eco47III sites, one of which must be eliminated, using, forexample, site-directed mutagenesis. The remaining Eco47III site, locatedupstream of exon 0, is used to linearize the vector.

Transfection of Reporter Constructs: The linearized recombination vectorcontaining the reporter gene and marker gene is introduced into culturedcells, including human cells, by any of the various transfection methodsknown in the art. For example, the linearized vector can be transfectedusing the methods described in Example 15. The linearized recombinationvector contains ABC1 sequences which allow the vector to integrate intothe cellular genome at the site of the endogenous ABC1 gene. Theaddition of an appropriate antibiotic to the culture medium allows theselection of only those cells in which the reporter gene and marker genehave integrated downstream of the endogenous ABC1 promoter in the properorientation. For instance, if the vector contains a puromycin resistancegene inserted downstream of the reporter gene, the transfected cellsshould be grown in the presence of puromycin. Only those cells that havea properly integrated puromycin resistance gene, and can thereby encodea functional protein, will survive in the presence of puromycin. Thus,the transfected cells should be grown under conditions that induce ABC1promoter activity in the presence of the appropriate antibiotic.Surviving cells can be clonally cultured and the DNA sequenced using PCRor southern blot analysis to test for proper integration of genomicsequences.

The resultant cells containing a reporter gene under the control of theendogenous ABC1 promoter can be used to determine whether a testcompound modulates the expression of ABC1. The ABC1 modulating activityof a compound is determined by assaying the level of reporter geneexpression found in the cells exposed to the test compound. For example,cells having an integrated luciferase gene can be used to determine theABC1 modulating activity of a test compound by measuring the amount ofluciferase activity found in cells exposed to the compound.

Example 17

This example demonstrates that ligands for nuclear receptors up-regulatethe expression of a reporter gene under the control of the ABC1promoter.

To determine whether ligands for the LXRα, LXRβ and RXRα nuclearreceptors could regulate ABC1 gene expression, the pAPR1 plasmidcontaining the luciferase reporter gene under control of the ABC1promoter was transfected into RAW 264.7 cells treated with at least oneligand for the nuclear receptors (FIG. 12).

Construction and Transfection of Reporter Constructs: Reporter constructpAPR1 and control reporter construct pGL3-Basic were obtained asdescribed in Example 15. RAW 264.7 cells were maintained in culture andtransfected with either pGL3-Basic (1 μg) or pAPR1 (1 μg) as describedin Example 15. The transfected RAW 264.7 cells were treated with eitherethanol (EtOH) (0.1% v/v), 20(S)-hydroxycholesterol (20(S)OH-chol)(10M), 9-cis retinoic acid (9-cis RA) (10 μM) or both 20(S)OH-chol and9-cis RA (20 μM total) for 24 hours. The luciferase activity wasmeasured and reported as relative light units as described in Example15.

Results: The results of this study are shown in FIG. 12. Control cellstransfected with pGL3-Basic showed no luciferase activity (data notshown). Cells transfected with pAPR1 produced a 19-fold increase inluciferase reporter activity in the presence of 200H-chol, a 16-foldincrease in luciferase activity in the presence of 9-cis RA, and a280-fold increase in luciferase activity in the presence of both ligandscompared with EtOH control. These results indicate that both the steroland retinoid elicit a strong transcription response from the ABC1 5′flanking sequence in pAPR1. Further, there is an apparent synergisticeffect of the two classes of compounds, as can be seen by the dramaticincrease in luciferase activity found in cells treated with bothligands. It is known that LXRα and RXRα receptors form activeheterodimers. Thus, the ligand-induced activation of both nuclearreceptors simultaneously could produce the observed synergistic increasein transcription.

These data demonstrate that hydroxy sterols, such as 20(S)hydroxycholesterol, and retinoids, such as 9-cis retinoic acid, activatethe ABC1 promoter, indicating that these and related compounds can beuseful in the development of therapeutic compounds to increase ABC1expression in macrophage cells to rid peripheral sites of excesscholesterol. Additionally, the present ABC1 promoter/reporter genescreening assay can be used to screen other compounds that increase ABC1expression to identify further therapeutic compounds.

Example 18

This example demonstrates the further characterization of the ABC1promoter region, including the identification of an LXR responseelement.

To determine which portion of the 5′ flanking region of ABC1 retainstranscriptional activity in response to nuclear ligands, variousplasmids containing a different portion of the 5′ flanking region and aluciferase reporter gene were transfected into RAW 264.7 cells treatedwith at least one ligand for the nuclear receptors. Using this system, asterol response element corresponding to nucleotides 1480-1510 of SEQ IDNO: 3 was identified. The sterol response element contains a directrepeat-4 element TGACCGatagTAACCT (SEQ ID NO: 61). Confirmation of thesterol response element was obtained using site-directed mutagenesis andband-shift assay techniques.

Construction of Reporter Constructs: Reporter construct pAPR1 andcontrol reporter construct pGL3-Basic were obtained as described inExample 15. Reporter constructs containing either nucleotides 1-1532,1080-1643, 1181-1643, 1292-1643, or 1394-1643 of SEQ ID NO: 3 were alsoconstructed. A reporter construct containing nucleotides 1080-1643 ofSEQ ID NO: 3 (GL-6a) was constructed as described in Example 15. Areporter construct containing nucleotides 1-1532 of SEQ ID NO: 3 wasconstructed by digestion of pAPR1 with Spe I and Nhe I, and re-ligationof the gel-purified vector fragment. A reporter construct containingnucleotides 1181 to 1643 was constructed by firstly digesting GL-6a withSty I, blunting the cohesive ends with Klenow enzyme, digesting theresultant vector with Sac I, and isolating the 462 base pair blunt-Sac Icohesive end fragment. This was cloned into a vector obtained bydigestion of GL-6a with Acc65 I, blunting of the cohesive ends withKlenow enzyme, digestion with Sac I and gel isolation of the vectorfragment. A reporter construct containing nucleotides 1292-1643 wasconstructed by consecutive digestion of GL-6a with Acc65 I, blunting theends with Klenow enzyme, digestion with Sac II, blunting the ends withT4 polymerase, and re-ligation of the gel-isolated vector fragment. Areporter construct containing nucleotides 1394-1643 was constructed bydigestion of GL-6a with Acc65 I, blunting the ends with Klenow enzyme,subsequent digestion with Apa I, end-blunting with T4 polymerase andre-ligation of the gel-isolated vector fragment.

Transfection of Reporter Constructs: The RAW 264.7 cells were maintainedin culture and transfected with either pGL3-Basic (1 μg), pAPR1 (1 μg),or one of the other reporter constructs according to the methoddescribed in Example 15. The transfected RAW 264.7 cells were treatedwith either ethanol (EtOH) (0.1% v/v), 20(S)-hydroxycholesterol(20(S)OH-chol) (10 μM), 22(R)-hydroxycholesterol (22(R)OH-chol) (10 μM),9-cis retinoic acid (9-cis RA) (10 μM), or both 20(S)OH-chol and 9-cisRA (20 μM total) for 24 hours. The luciferase activity was measured andreported as relative light units as described in Example 15.

Site-Directed Mutagenesis: The sterol response element corresponding tonucleotides 1480-1510 of SEQ ID NO: 3 was mutated in the 1080-1643sequence described above using site-directed mutagenesis. Specifically,the response element containing a direct repeat-4 elementTGACCGatagTAACCT (SEQ ID NO: 61) was mutated to CTGCACatagTAACCT (SEQ IDNO: 62) using the GeneEditor system (Promega, Madison, Wis.) accordingto the manufacturer's protocol.

Gel-Shift Assays: Nuclear extract was prepared from RAW 264.7 cells bythe method of Ohlsson et al., Cell, 45:35-44 (1986). ³²P-labeledoligonucleotides (5 ng) corresponding to the LXR response element(TCGAGTGACCGATAGTAACCTCTCGA; SEQ ID NO: 56) and its mutated counterpart(TCGAGCTGCACATAGTAACCTCTCGA; SEQ ID NO: 57) were individually incubatedwith 5 μg of nuclear protein for 30 minutes at room temperature in 20 mMHEPES, pH 7.9, 60 mM KCL, 1 mM MgCl₂, 1 mM DTT, 66.6 μg/ml poly(dIdC),and 10% glycerol in the presence or absence of 1 μg antiserum to LXRαand LXRβ (Santa Cruz Biotechnology, Cat. No. SC-1591, Santa Cruz,Calif.) or antiserum to RXR (Santa Cruz Biotechnology, Cat. No. SC-774,Santa Cruz, Calif.). The protein-DNA complexes were applied to a 4%non-denaturing polyacrylamide gel for 1.5 hours at 150V in 0.5× TBEbuffer. The protein-DNA complexes were detected by autoradiography ofthe dried gel.

Results: Transfection with the individual reporter constructs containingthe 5′ flanking region corresponding to nucleotides 1-1643 (i.e.,pAPR1), 1-1532, 1080-1643, 1181-1643, 1292-1643, or 1394-1643 of SEQ IDNO: 3 each produced the same results. All of the individual constructsproduced a 3 to 4-fold increase in luciferase reporter activity in thepresence of 20 (S)OH-chol or 22 (R)OH-chol compared with EtOH control.Also, all of the individual constructs produced an 8 to 10-fold increaseluciferase reporter activity in the presence of 9-cis RA. In addition,transfection with any of the constructs produced a 25 to 50-foldincrease in luciferase activity in the presence of oxysterol ligand(either (20 (S)OH-chol or 22(R)OH-chol)) and retinoid ligand (9-cis RA)together compared with EtOH control, indicating a synergisticinteraction. Each of the described constructs demonstrated comparablelevels of luciferase activity in response to the ligands tested,indicating that even the shorter 5′ flanking sequences containedtranscriptional regulatory sequences for sterols and retinoids.Specifically, these results demonstrated that the transcriptionalregulatory sequences for sterols and retinoids are located in the 5′flanking region corresponding to nucleotides 1394-1532 of SEQ ID NO: 3.

These results were confirmed by luciferase assays using a reporterconstruct containing the wild-type sequence corresponding to nucleotides1080-1643 of SEQ ID NO: 3 and a reporter construct containing a mutatedsequence corresponding to nucleotides 1080-1643 of SEQ ID NO: 3, whereinthe sterol response element found at nucleotides 1480-1500 was mutatedas described above. Transfection with the wild-type sequence produced atranscriptional response, as measured by an increase in luciferaseactivity, in the presence of either 20 (S)OH-chol or 9-cis RA alone andproduced a synergistic response in the presence of both ligandstogether. In contrast, transfection with the mutated sequence did notproduce a transcriptional response in the presence of 20 (S)OH-chol or22 (R)OH-chol. Transfection of the mutated sequence preserved a reducedresponse to 9-cis RA, producing a 4 to 5-fold increase intranscriptional activity, rather than the 8 to 10-fold increase observedwith the wild-type sequence. Transfection of the mutated sequence alsoabolished the synergistic transcriptional response seen in the presenceof 20 (S)OH-chol and 9-cis RA together. These results were furtherconfirmed by gel-shift assays using the sterol consensus sequence(nucleotides 1480-1510) and its mutated counterpart. The gel-shiftassays showed that while nuclear binding proteins isolated from RAW264.7 cells bound to the sterol consensus sequence, nuclear proteins didnot bind to the mutated sequence. Furthermore, incubation of nuclearproteins with the wild-type sterol consensus sequence in the presence ofLXR antiserum resulted in the formation of supershifted complexes (i.e.antibody-protein-DNA complexes), identifying the sequence as a sterolresponse element that binds nuclear receptor LXR. In contrast,incubation of nuclear proteins with the wild-type sterol responseelement in the presence of RXR antiserum did not result in the formationof supershifted complexes, indicating that RXR does not bind to thissequence. These results show that the mutation which destroys nuclearprotein binding to the consensus sequence also abolishes thetranscriptional response to LXR ligands and diminishes the response toRXR ligands. Furthermore, the nuclear binding studies performed in thepresence of LXR or RXR antiserum confirmed that the consensus sequencefound at nucleotides 1480-1510 is an LXR response element. This elementalso mediates a partial response to 9-cis RA.

All of the references cited herein, including patents and publications,are hereby incorporated in their entireties by reference. While theinvention has been described with an emphasis upon preferred aspects ofthe invention, it will be readily apparent to those of ordinary skill inthe art that variations of the preferred embodiments can be used andthat it is intended that the invention can be practiced otherwise thanis specifically described herein. Accordingly, the present inventionincludes all modifications encompassed within the spirit and scope ofthe invention as defined by the following claims.

1. A kit suitable for screening a compound to determine the ABC1expression modulating activity of the compound comprising a recombinantreporter construct comprising a reporter cDNA operatively linked to anexpression modulating portion of the mammalian ABC1 gene in an amountsufficient for at least one assay and instructions for use.
 2. The kitof claim 1, wherein the expression modulating portion of the mammalianABC1 gene comprises SEQ ID NO:
 3. 3. The kit of claim 1, wherein theexpression modulating portion of the mammalian ABC1 gene comprisesnucleotides 1-1532, 1080-1643, 1181-1643, 1292-1643, 1394-1643, or1394-1532 of SEQ ID NO:
 3. 4. The kit of claim 1, wherein the reportercDNA is selected from the group consisting of luciferase,-galactosidase, chloramphenicol acetyl transferase, and greenfluorescents protein cDNA.
 5. The kit of claim 2, wherein the reportercDNA is a luciferase cDNA.
 6. The kit of claim 3, wherein the reportercDNA is a luciferase cDNA.
 7. The kit of claim 5, wherein therecombinant reporter construct is pAPR1.
 8. The kit of claim 1, furthercomprising means for detecting the reporter gene.